CN104645416A - In vitro construction method for tissue engineering human corneal stroma - Google Patents
In vitro construction method for tissue engineering human corneal stroma Download PDFInfo
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- CN104645416A CN104645416A CN201510039441.7A CN201510039441A CN104645416A CN 104645416 A CN104645416 A CN 104645416A CN 201510039441 A CN201510039441 A CN 201510039441A CN 104645416 A CN104645416 A CN 104645416A
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Abstract
The invention relates to an in vitro construction method for tissue engineering human corneal stroma. The in vitro construction method comprises the following steps: preparing cell suspension from human corneal stroma cells with a special in vitro construction culture solution A of tissue engineering human corneal stroma; injecting the cell suspension into an acellular porcine corneal matrix carrier bracket which is rehydrated by a special rehydration solution for the carrier bracket, and putting the bracket into a 24-pore culture plate; and adding the special in vitro construction culture solution A, carrying out in vitro culture, and replacing a special in vitro construction culture solution B, so as to obtain the tissue engineering human corneal stroma. The in vitro construction method is low in production cost, and short in elapsed time; the integrity of the carrier bracket structure can be well kept; the constructed tissue engineering human corneal stroma is similar to normal human corneal stroma in morphological structure and transparency, and has the functions of the normal human corneal stroma, and can be applied to repairing of corneal stroma abnormity as a substitution for donating cornea, so that the in vitro construction method can be applied to mass production and meet the high-quality requirements of the tissue engineering human corneal stroma.
Description
Technical field
The invention belongs to tissue engineering comea substrate constructing technology field, be specifically related to a kind of vitro construction method of organizational project people corneal stroma.
Background technology
People's corneal stroma is made up of people's keratocyte and 200-250 collagen fiber flaggy, in maintenance corneal transparency and corneal thickness etc., have irreplaceable effect.Cornea once be infected, degree of depth thermal burn, degree of depth chemical burn or operation wound etc., people's keratocyte generation damage in various degree will be caused, and then cause corneal edema and cicatrization and make visual impairment, severe patient will cause cornea opaque, namely cause keratopathy blind disease, common are the muddy and reticular corneal of collagen speckle, subepithelial stroma muddy etc.At present, the transplanting of people's corneal stroma is the unique channel of the sick blind healing of current corneal stroma, about there are nearly 5,000,000 people of the blind patient of keratopathy in China, although they can be cured by corneal transplantation, cause most patient cannot recover lost eyesight because can not get the donor that is suitable for owing to contributing the serious scarcity of cornea.Since eighties of last century, the fast development of cornea histoengineering new branch of science, the external structure of organizational project people corneal stroma is made to become possibility, also be solve a kind of new feasible way contributing cornea quantity wretched insufficiency at present, the sick blind patient of more various corneal stroma brings new hope by the clinical transplantation of organizational project people corneal stroma with treatment.
But in the external structure research of organizational project people corneal stroma, how to obtain enough people's keratocytes as seed cell, desirable carrier bracket, and external construction method, be the study hotspot of current organizational project people corneal stroma external structure.Chinese scholars utilize immortal human keratocyte (Peters DM etc., 1996 of Oncogene Transfection respectively, Doillon CJ etc., 2003, Reichl S etc., 2004, Manzer AK etc., 2009, Yoshiko K etc., 2010), people's keratocyte (Barbaro V etc., 2009 of original cuiture, Proulx S etc., 2010) and people's keratocyte (Crabb RA etc., 2005 in successive transfer culture 3-8 generation, Builles N etc., 2007, VranaE etc., 2008) as seed cell, utilize collagen protein-chondroitin sulfate gel (Griffith M etc. respectively, 1999), collagen protein-poly-N-isopropyl acrylamide polymer blend (Shimmura S etc., 2003), collagen protein-glycosaminoglycans-chitosan foam (Builles N etc., 2007), polyglycolic acid does not disassemble fiber (Liu and Cao, 2007), hydrogel (Mimura T etc., 2008), transplant with hollow man corneal film (Barbaro V etc., 2009), multilamellar collagen fiber (Builles N etc., 2010), extracellular matrix (the Proulx S etc. of people's keratocyte secretion, 2010) and de-cell porcine cornea substrate (Yoeruek E etc., 2011) etc. as carrier bracket, construct people's corneal stromal tissue analog that form and organizational structure and normal cornea substrate are approximate in vitro, for the external structure of organizational project people corneal stroma opens road, but owing to inoculating method many employings direct inoculation of seed cell, cell is caused to occur contact inhibition phenomenon because density is excessive, and the method exits cultivate in vitro chronic, moreover, the immortalized cells of seed cell used or transfection oncogene has potential oncogenicity, or from eye bank's cornea primary cultured cell or only pass the successive transfer culture cell in 3-8 generation, the quantity of the people's keratocyte namely obtained is very limited, thus greatly limit scale external structure and the clinical practice thereof of organizational project people corneal stroma.The research report of existing organizational project people corneal stroma external structure, is still confined to experimentation, cannot meet the sick blind clinical wilderness demand of numerous corneal stroma at all.
2011, Fan Tingjun etc. successfully established non-transfection, people's keratocyte system without oncogenicity, can be the scale of organizational project people corneal stroma and built the seed cell source providing enough.2014, Fan Tingjun etc. carry out hypotonic swelling to the fresh pig corneal film cut, after multigelation smudge cells, with DNA-RNA ferment treatment removal residual nucleic acid substances wherein, then the special cross-linking agent solution of cornea containing quality percent by volume 10%-20% dextran solution and quality percent by volume 0.1%-0.2% riboflavin is immersed, ultraviolet light A is successively utilized to irradiate crosslinked, rinsing, air-dry and ionizing radiation sterilizing, it is transparent good successfully to prepare, mechanicalness is strong, the tissue engineering comea carrier bracket (hereinafter referred to as carrier bracket) desirable with people's cornea seed cell biocompatibility---de-cell porcine cornea substrate, successfully solve the scale of organizational project people corneal stroma and build the problem lacking a large amount of ideal carrier support.Therefore, setting up a kind of method utilizing people's keratocyte and carrier bracket external structure organizational project people corneal stroma fast as early as possible, is that the sick blind key of whole world corneal stroma is benefited in the large-scale production of tissue engineering comea substrate and clinical practice.
Summary of the invention
The object of the invention is to be seed cell with people's keratocyte, to take off cell porcine cornea substrate for carrier bracket, provide a kind of vitro construction method of organizational project people corneal stroma, to make up the deficiencies in the prior art.
Vitro construction method of the present invention comprises:
By organizational project people corneal stroma external structure special culture solution A (hereinafter referred to as external structure special culture solution A), people's keratocyte is made cell suspension, again this pallium cell injection to be entered with making in the round carrier support of required size with corneal trephine after the process of carrier bracket special complex aqueous solution, be placed in 24 well culture plate holes, after adding external structure special culture solution A, be placed in 37 DEG C, In vitro culture is carried out in 5% CO2 gas incubator, change organizational project people corneal stroma external structure special culture solution B (hereinafter referred to as external structure special culture solution B) in incubation to continue to cultivate, namely the organizational project people corneal stroma that organizational structure is close with Corneal substrate with function is obtained.
The preparation method of above-mentioned people's keratocyte suspension: first get people's keratocyte, logarithmic growth after date is cultured to the DMEM/F12 culture fluid containing 15%-20% calf serum, again with 0.25% trypsin solution carry out digesting, centrifugal collecting cell precipitation, last again by external structure special culture solution A re-suspended cell precipitation, obtain people's keratocyte suspension of homogenizing.
The cell concentration range of above-mentioned people's keratocyte suspension is 5.0 × 10
6-1.0 × 10
7individual cells/ml.
The method of above-mentioned In vitro culture: draw above-mentioned people's keratocyte suspension with microsyringe, each carrier bracket evenly injects 4-6 pin, every pin 1-2 microlitre, insert in 24 well culture plate holes, then add external structure special culture solution A, be placed in 37 DEG C, 5% CO2 gas incubator carries out In vitro culture, change external structure special culture solution B once every 24-36 hour, cultivate after 72-120 hour, obtain organizational project people corneal stroma.
The formula of above-mentioned external structure special culture solution A: the DMEM/F12 culture fluid containing 15% calf serum and 0.3 ‰-0.5 ‰ tranamic acid.
The formula of above-mentioned external structure special culture solution A with the addition of again 0.2 ‰-0.4 ‰ fibronectins.
The formula of above-mentioned external structure special culture solution B: the DMEM/F12 culture fluid containing 15% calf serum and 0.3 ‰-0.5 ‰ tranamic acid.
The formula of above-mentioned external structure special culture solution B with the addition of again 0.1 ‰-0.5 ‰ ε-lpsilons.
The formula of the special complex aqueous solution of above-mentioned carrier bracket: the DMEM/F12 culture fluid containing 1% tobramycin.
Seed cell in the present invention is the normal and normal people's keratocyte of structure function from the caryogram of people's keratocyte system of non-transfection, this cell growth state is good, without any tumorigenesis risk, the batch external structure being organizational project people corneal stroma by continuous passage provides enough seed cells; The preparation technology of carrier bracket only uses physics (hypotonic, freeze thawing, air-dry) and enzyme (DNA-RNA enzyme) process, do not use any poisonous chemical reagent and medicine, safety is high, antigenicity is little, carrier bracket after rehydration has that the transparency is good, compact structure is complete, mechanicalness is strong, and people's cornea seed cell good biocompatibility, be easy to the features such as seed cell adheres to, moves and grows, production cost is low, meets the needs of organizational project people corneal stroma a large amount of carrier bracket used.The present invention inoculates seed cell and adopts injection inoculation method, not only substantially reduces the time of In vitro culture, and better can keep the integrity of carrier bracket structure compared with direct inoculation.
Present invention process is scientific and reasonable, constructed by the organizational project people corneal stroma that goes out similar to Corneal substrate on morphosis with transparency, and there is the function of normal cornea substrate, be transplanted to new zealand rabbit, its cornea can be made to continue to keep transparent difference 150 days in beasle dog and Rhesus Monkeys ' eyes, more than 820 days and 750 days, show that the organizational project people corneal stroma gone out constructed by the present invention can be used as the reparation of substitute for corneal stroma exception of donation cornea, therefore can be used for producing the high-quality demand met organizational project people corneal stroma in batches, and the cost of organizational project people corneal stroma external structure is low.
Detailed description of the invention
The compound method of the various solution that the present invention relates to:
1, the preparation of external structure special culture solution A: the DMEM/F12 culture fluid 80 milliliters getting normal compound, add tranamic acid 30-50 milligram, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add 15 milliliters of calf serums, add above-mentioned DMEM/F12 culture fluid to 100 milliliter, be external structure special culture solution A;
In order to improve the adhesion effect of people's keratocyte in carrier bracket, the formula of above-mentioned culture fluid will with the addition of the fibronectin of 20-40 milligram again;
2, the preparation of external structure special culture solution B: the DMEM/F12 culture fluid 80 milliliters getting normal compound, add tranamic acid 30-50 milligram, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add 15 milliliters of calf serums, add above-mentioned DMEM/F12 culture fluid to 100 milliliter, be external structure special culture solution B;
In order to improve the attachment of people's keratocyte in carrier bracket and migration effect, the formula of above-mentioned culture fluid will with the addition of the ε-lpsilon of 10-50 milligram again;
3, the preparation of the special complex aqueous solution of carrier bracket: the DMEM/F12 culture fluid 80 milliliters getting normal compound, add tobramycin 1 gram, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add above-mentioned DMEM/F12 culture fluid to 100 milliliter, be the special complex aqueous solution of carrier bracket.
Specific embodiment of the invention step:
1, the preparation of people's corneal stroma seed cell: fetch from non-transfection, normal and the normal people's keratocyte of structure function of caryogram without oncogenicity people corneal stroma system, amplification cultivation is carried out mid-37 DEG C of floor space 75 square centimeter culture bottle with the DMEM/F12 culture fluid containing 15%-20% calf serum, cell proliferation 36-48 is little after exponential phase, with glass dropper sucking-off culture fluid, add 0.25% trypsin solution digestion 1-2 minute, the old culture fluid adding sucking-off before this stops digestion, 1000-1500 rev/min of centrifugal 10-15 minute, cell precipitation suspends with 4 milliliters of external structure special culture solution A and evenly makes people's keratocyte suspension of homogenizing, after utilizing CASY cell counter to carry out cell counting, adjust cell concentration to 5.0 × 10 with above-mentioned external structure special culture solution A
6-1.0 × 10
7individual cells/ml,
2, the preparation of carrier bracket: after utilizing the special complex aqueous solution of carrier bracket to carry out rehydration process in 1-2 hour to carrier bracket immersion, then with corneal trephine, the carrier bracket after rehydration is broken into the disk of required size;
3, the external structure of organizational project people corneal stroma: draw above-mentioned people's keratocyte suspension with microsyringe, each carrier bracket evenly injects 4-6 pin, every pin 1-2 microlitre; Carrier bracket after injection cell suspension is put into 24 well culture plates, add above-mentioned external structure special culture solution A to 1 milliliter, put 37 DEG C, cultivate in 5% CO2 gas incubator, external structure special culture solution B is changed once every 24-36 hour, continue to cultivate after 72-120 hour, be the organizational project people corneal stroma that external structure goes out.
The present invention is illustrated further in conjunction with multiple embodiments below:
Embodiment 1
First get the DMEM/F12 culture fluid 80 milliliters of normal compound, add tranamic acid 30 milligrams, fibronectin 20 milligrams, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add 15 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, make external structure special culture solution A;
Get people's keratocyte again, amplification cultivation is carried out mid-37 DEG C of floor space 75 square centimeter culture bottle with the DMEM/F12 culture fluid containing 15% calf serum, cell proliferation 48 is little after exponential phase, with glass dropper sucking-off culture fluid, add 0.25% trypsin solution and digest 1.5 minutes, the old culture fluid adding sucking-off before this stops digestion, after 1500 revs/min of centrifugal 10 minutes collecting cells precipitation, above-mentioned cell precipitation to be suspended even adult cornea's stromal cell suspension with 4 milliliters of above-mentioned external structure special culture solution A; After utilizing CASY cell counter to carry out cell counting, adjust cell concentration to 1.0 × 10 with above-mentioned external structure special culture solution A
7individual cells/ml;
Then get the DMEM/F12 culture fluid 80 milliliters of normal compound, add tobramycin 1 gram, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add above-mentioned DMEM/F12 culture fluid to 100 milliliter, be the special complex aqueous solution of carrier bracket.
After carrier bracket being soaked and carries out rehydration process in 2 hours with carrier bracket special complex aqueous solution again, with corneal trephine, the carrier bracket after rehydration is broken into the disk of diameter 6.5 millimeters again, above-mentioned people's keratocyte suspension is drawn with microsyringe, each carrier bracket evenly injects 6 pins, every pin 1 microlitre; Then the carrier bracket after injection cell suspension put into 24 well culture plates, add above-mentioned external structure special culture solution A to 1 milliliter, put 37 DEG C, carry out In vitro culture in 5% CO2 gas incubator;
Get the DMEM/F12 culture fluid 80 milliliters of normal compound again, add tranamic acid 30 milligrams, ε-the lpsilon of 10 milligrams, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add 15 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, make external structure special culture solution B; Finally changed an external structure special culture solution B every 36 hours, continue cultivation 120 hours, be the organizational project people corneal stroma of external structure.
Embodiment 2
First get the DMEM/F12 culture fluid 80 milliliters of normal compound, add tranamic acid 40 milligrams, fibronectin 30 milligrams, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add 15 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, make external structure special culture solution A;
Get people's keratocyte again, amplification cultivation is carried out mid-37 DEG C of floor space 75 square centimeter culture bottle with the DMEM/F12 culture fluid containing 20% calf serum, cell proliferation 36 is little after exponential phase, with glass dropper sucking-off culture fluid, add 0.25% trypsin solution and digest 2 minutes, the old culture fluid adding sucking-off before this stops digestion, and 1000 revs/min centrifugal 15 minutes, and cell precipitation suspends with 4 milliliters of above-mentioned external structure special culture solution A and evenly makes people's keratocyte suspension; After utilizing CASY cell counter to carry out cell counting, adjust cell concentration to 7.0 × 10 with above-mentioned external structure special culture solution A
6individual cells/ml;
Then get the DMEM/F12 culture fluid 80 milliliters of normal compound, add tobramycin 1 gram, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add above-mentioned DMEM/F12 culture fluid to 100 milliliter, be the special complex aqueous solution of carrier bracket.
After carrier bracket being soaked and carries out rehydration process in 1.5 hours with carrier bracket special complex aqueous solution again, with corneal trephine, the carrier bracket after rehydration is broken into the disk of diameter 8.0 millimeters again, above-mentioned people's keratocyte suspension is drawn with microsyringe, each carrier bracket evenly injects 5 pins, every pin 1.5 microlitre; Then the carrier bracket after injection cell suspension put into 24 well culture plates, add above-mentioned external structure special culture solution A to 1 milliliter, put 37 DEG C, carry out In vitro culture in 5% CO2 gas incubator;
Get the DMEM/F12 culture fluid 80 milliliters of normal compound again, add tranamic acid 40 milligrams, ε-the lpsilon of 30 milligrams, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add 15 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, make external structure special culture solution B; Finally changed an external structure special culture solution B every 30 hours, continue cultivation 96 hours, be the organizational project people corneal stroma of external structure.
Embodiment 3
First get the DMEM/F12 culture fluid 80 milliliters of normal compound, add tranamic acid 50 milligrams, it is degerming with the filtering with microporous membrane of 0.22 micron after fibronectin 40 milligrams dissolves completely, add 15 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, make external structure special culture solution A;
Get people's keratocyte again, amplification cultivation is carried out mid-37 DEG C of floor space 75 square centimeter culture bottle with the DMEM/F12 culture fluid containing 15% calf serum, cell proliferation 48 is little after exponential phase, with glass dropper sucking-off culture fluid, add 0.25% trypsin solution and digest 1 minute, the old culture fluid adding sucking-off before this stops digestion, and 1200 revs/min centrifugal 12 minutes, and cell precipitation suspends with 4 milliliters of above-mentioned external structure special culture solution A and evenly makes people's keratocyte suspension; After utilizing CASY cell counter to carry out cell counting, adjust cell concentration to 8.0 × 10 with above-mentioned external structure special culture solution A
6cells/ml;
Then get the DMEM/F12 culture fluid 80 milliliters of normal compound, add tobramycin 1 gram, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add above-mentioned DMEM/F12 culture fluid to 100 milliliter, be the special complex aqueous solution of carrier bracket.
After carrier bracket being soaked and carries out rehydration process in 1 hour with carrier bracket special complex aqueous solution again, with corneal trephine, the carrier bracket after rehydration is broken into the disk of diameter 9.5 millimeters again, above-mentioned people's keratocyte suspension is drawn with microsyringe, each carrier bracket evenly injects 4 pins, every pin 2 microlitre; Then the carrier bracket after injection cell suspension put into 24 well culture plates, add above-mentioned external structure special culture solution A to 1 milliliter, put 37 DEG C, carry out In vitro culture in 5% CO2 gas incubator;
Get the DMEM/F12 culture fluid 80 milliliters of normal compound again, add tranamic acid 50 milligrams, ε-the lpsilon of 50 milligrams, degerming with the filtering with microporous membrane of 0.22 micron after dissolving completely, add 15 milliliters of calf serums, add the DMEM/F12 culture fluid to 100 milliliter of normal compound, make external structure special culture solution B; Finally changed an external structure special culture solution B every 24 hours, continue cultivation 72 hours, obtain the organizational project people corneal stroma of external structure.
As can be seen here, utilize the organizational project people corneal stroma of method external structure of the present invention, the substitute that can be used as people's cornea is applied to the reparation of corneal stroma exception, and easily accomplishes scale production.
Claims (10)
1. the vitro construction method of an organizational project people corneal stroma, it is characterized in that, with organizational project people corneal stroma external structure special culture solution A, people's keratocyte is made cell suspension, again this pallium cell injection is entered in the de-cell porcine cornea medium carrier support after carrier bracket complex aqueous solution rehydration, be placed in 24 well culture plate holes, after adding external structure special culture solution A, be placed in 37 DEG C, In vitro culture is carried out in 5% CO2 gas incubator, period is changed external structure special culture solution B and continues to cultivate, namely the organizational project people corneal stroma that organizational structure is close with Corneal substrate with function is obtained.
2. the vitro construction method of organizational project people corneal stroma as claimed in claim 1, it is characterized in that the preparation method of above-mentioned people's keratocyte suspension: first get people's keratocyte, logarithmic growth after date is cultured to the DMEM/F12 culture fluid containing 15%-20% calf serum, again with 0.25% trypsin solution carry out digesting, centrifugal collecting cell precipitation, last again by external structure special culture solution A re-suspended cell precipitation, obtain people's keratocyte suspension of homogenizing.
3. the vitro construction method of organizational project people corneal stroma as claimed in claim 1, it is characterized in that the preparation method of above-mentioned carrier bracket: first cut fresh pig corneal film and carry out hypotonic swelling, after multigelation smudge cells, with DNA-RNA ferment treatment removal residual nucleic acid substances wherein, then the special cross-linking agent solution of cornea containing quality percent by volume 10%-20% dextran solution and quality percent by volume 0.1%-0.2% riboflavin is immersed, ultraviolet light A is successively utilized to irradiate crosslinked, rinsing, air-dry and ionizing radiation sterilizing, finally use carrier bracket complex aqueous solution to after carrier bracket rehydration, the round carrier support of required size is made with corneal trephine.
4. the vitro construction method of organizational project people corneal stroma as claimed in claim 1, it is characterized in that the method for above-mentioned In vitro culture: draw above-mentioned people's keratocyte suspension with microsyringe, each carrier bracket evenly injects 4-6 pin, every pin 1-2 microlitre, then external structure special culture solution A is added, be placed in 37 DEG C, 5% CO2 gas incubator carries out In vitro culture, external structure special culture solution B is changed once every 24-36 hour, cultivate after 72-120 hour, obtain organizational project people corneal stroma.
5. the vitro construction method of the organizational project people corneal stroma as described in claim 1 and 2, is characterized in that the formula of above-mentioned external structure special culture solution A: the DMEM/F12 culture fluid containing 15% calf serum and 0.3 ‰-0.5 ‰ tranamic acid.
6. the vitro construction method of the organizational project people corneal stroma as described in claim 1 and 2, is characterized in that the formula of above-mentioned external structure special culture solution A with the addition of again 0.2 ‰-0.4 ‰ fibronectins.
7. the vitro construction method of the organizational project people corneal stroma as described in claim 1 and 4, is characterized in that the formula of above-mentioned external structure special culture solution B: the DMEM/F12 culture fluid containing 15% calf serum and 0.3 ‰-0.5 ‰ tranamic acid.
8. the vitro construction method of the organizational project people corneal stroma as described in claim 1 and 4, is characterized in that the formula of above-mentioned external structure special culture solution B with the addition of again 0.1 ‰-0.5 ‰ ε-lpsilons.
9. the vitro construction method of the organizational project people corneal stroma as described in claim 1 and 3, is characterized in that the formula of above-mentioned carrier bracket complex aqueous solution: the DMEM/F12 culture fluid containing 1% tobramycin.
10. the organizational project people corneal stroma constructed by claim 1, is characterized in that the reparation being applied to corneal stroma exception as the substitute contributing people's cornea.
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| CN111110921A (en) * | 2019-12-23 | 2020-05-08 | 中国海洋大学 | In-vitro construction method of tissue engineering posterior lamella cornea |
| CN115006593A (en) * | 2022-06-26 | 2022-09-06 | 中国海洋大学 | In-vitro construction method of mechanical enhanced tissue engineering corneal endothelium |
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| CN115006593A (en) * | 2022-06-26 | 2022-09-06 | 中国海洋大学 | In-vitro construction method of mechanical enhanced tissue engineering corneal endothelium |
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