CN106955372A - A kind of construction method of tissue engineering comea endothelium - Google Patents
A kind of construction method of tissue engineering comea endothelium Download PDFInfo
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- CN106955372A CN106955372A CN201710234952.3A CN201710234952A CN106955372A CN 106955372 A CN106955372 A CN 106955372A CN 201710234952 A CN201710234952 A CN 201710234952A CN 106955372 A CN106955372 A CN 106955372A
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Abstract
The invention provides a kind of tissue engineering comea endothelium and its construction method, including prepare the steps such as porcine cornea descemet's membrane, culture, induction human pluripotent stem cells, structure tissue engineering comea endothelium.The construction method and its tissue engineering comea endothelium of the tissue engineering comea endothelium of the present invention, compared to amnion or other carriers, descemet's membrane used herein is cornea normal configuration in itself, will not be excessively soft, transparent good.Used seed cell is that human pluripotent stem cells induction is differentiated, and is not limited by primary cell multiplication capacity is low.Also, tissue engineering comea endothelium preparation process of the present invention is simple, wide material sources, it is easy to operate.
Description
Technical field
The invention belongs to field of tissue engineering technology, and in particular to a kind of construction method of tissue engineering comea endothelium.
Background technology
It is thin that corneal endothelium is attached to descemet's membrane (Descemet's membrane), the individual layer positioned at cornea most inner side
Born of the same parents, directly contact with aqueous humor, maintain corneal transparency by its active liquid pump function, keep cornea normal thickness.In adult cornea
Chrotoplast can not be regenerated, and impaired rear main divided a word with a hyphen at the end of a line by the extension of damage zone periphery cell is repaired.A variety of causes such as Fuch angles
Film endothelium is malnutritive, wound and operation etc. can cause endothelial cell to damage, when cell density less than 500-1500/
mm2When occur corneal endothelium function decompensation, cause corneal edema muddy, bleb type keratopathy be presented.For this kind of disease
Disease, can be treated by transplanting the method for endothelium merely, and the method clinically commonly used at present includes the angle with descemet's membrane
Skin grafting dermepenthesis and the endothelial cell transplanting with lamellar cornea in film, but the source of corneal donor or endothelial cell is deficient serious
Constrain the development of clinical such operation.
The content of the invention
Unexpectedly, it is found by the applicant that by by the human pluripotent stem cells combination porcine cornea descemet's membrane of suitable size, in vitro
It is to provide a kind of structure side of tissue engineering comea endothelium to build engineered corneal endothelium, i.e. the first object of the present invention
Method, comprises the following steps：
1) porcine cornea descemet's membrane is prepared：Porcine cornea is removed into porcine cornea endothelial cell, makes pig descemet's membrane and cornea base
Matter is completely separated, by the porcine cornea descemet's membrane completely separated dry, sterilize it is standby；
2) cultivate, induce human pluripotent stem cells：By the human pluripotent stem cells culture of cellar culture to suitable size, using luring
Culture medium induction is led, become big, cellular morphology to human pluripotent stem cells clone's inner cell volume is changed into polygon from circular or oval
Shape, arrangement is close, and clone's periphery cell terminates induction in fusiformis threadiness；The human pluripotent stem cells clone for terminating induction is disappeared
Use differential medium resuspended after change and count standby；
3) tissue engineering comea endothelium is built：By step 1) obtain porcine cornea descemet's membrane, add coating buffer wrapped
Quilt, then by step 2) human pluripotent stem cells after the induction differentiation that obtains are inoculated in step 1) the porcine cornea descemet's membrane that obtains,
After adhere-wall culture, change after fresh differential medium culture, obtain tissue engineering comea endothelium.
Preferably, in the construction method of tissue engineering comea endothelium of the present invention, the step 1) the pig angle that uses
Film builds the complete total corneal for planting piece diametric requirements or the porcine cornea for possessing descemet's membrane to meet.
Preferably, in the construction method of tissue engineering comea endothelium of the present invention, the step 1) described in dry
Sterilization treatment is using x ray irradiation x or adds antibiotic medicine sterilization treatment.
Preferably, in the construction method of tissue engineering comea endothelium of the present invention, the step 2) described in it is suitable
The size of the human pluripotent stem cells of size is 60-100 cell/clone.
Preferably, in the construction method of tissue engineering comea endothelium of the present invention, the inducing culture be
Following material is added on DMEM/F12 basal mediums to be made：Beta -mercaptoethanol, glutamine, Basic Fibroblast Growth Factor
(bFGF), nonessential amino acid (NEAA), serum substitute (knockout serum replacement, KSR), total trans dimension
Formic acid (RA)；Preferably, the beta -mercaptoethanol concentration is that 0.1mM, glutamine concentration are that 0.1mM, bFGF concentration are 4-
8ng/ml, NEAA be 20% (volume fraction) that 0.1mM, KSR concentration are total amount of liquid, RA concentration be 0.5-2 μM.
Preferably, in the construction method of tissue engineering comea endothelium of the present invention, the differential medium be containing
The 5%DKSFM culture mediums of ROCK inhibitor.
Preferably, in the construction method of tissue engineering comea endothelium of the present invention, the step 3) described in be coated with
Liquid is Laminin lens liquid or compound coating buffer (FNC Coating Mix).
It is highly preferred that in the construction method of tissue engineering comea endothelium of the present invention, comprising the following steps：
1) porcine cornea descemet's membrane is prepared：The porcine cornea that fresh or glycerine is preserved, uses the sterile physiological salt containing antibiotic
Water immersion, rinse 20 minutes after, endothelium is placed under Stereo microscope up, using cotton swab carefully wipe removal porcine cornea endothelium
Cell, porcine cornea is fixed using pincers, is first torn remnants iris root and exposure descemet's membrane free fraction off, is then used
Tack tweezer carefully grips the free place of pig descemet's membrane, and gently being torn to Central corneal makes pig descemet's membrane completely divide with corneal stroma
From.Operation should be noted dynamics, once descemet's membrane rupture curling can not be used.
It is enclosed within the porcine cornea descemet's membrane completely separated is smooth on internal diameter≤11.0mm antiseptic rubber annulus, interior surface
Upwards be attached at culture plate be allowed to dry, sterilizing, using it is preceding addition antibiotic immersion be placed in 37 DEG C, 5%CO2 incubators it is standby；
2) human pluripotent stem cells are induced：The human pluripotent stem cells culture of cellar culture to suitable size be begin to use containing
Visible human pluripotent stem cells clone expands and connected under RA inducing culture continuous induction 3-5 days, mirror, clones inner cell volume
Become big, cellular morphology and polygon is changed into from circular or oval, arrangement is close, clone's periphery cell is in fusiformis threadiness.Will be eventually
Use the 5%DKSFM differential mediums containing ROCK inhibitor resuspended after the human pluripotent stem cells Clone Digestion only induced and count
It is standby；
The human pluripotent stem cells clone of the suitable size refers to：About 60-100 cell under inverted phase contrast microscope/gram
It is grand, clone uniform in size, medium density；
The inducing culture containing RA is to add following material system on knockout DMEM/F12 basal mediums
Into：Beta -mercaptoethanol, glutamine, bFGF, NEAA, KSR, RA.After addition, beta -mercaptoethanol concentration is 0.1mM, glutamine
Concentration be 0.1mM, bFGF concentration be 4-8ng/ml, NEAA be 20% (volume fraction) that 0.1mM, KSR concentration are total amount of liquid,
RA concentration is 0.5-2 μM；
The 5%DKSFM differential mediums containing ROCK inhibitor be in DKSFM basal mediums add FBS and
What ROCK inhibitor was made.Its concentration be respectively 5% (volume fraction) of total amount of liquid, 5-20 μM；
3) tissue engineering comea endothelium is built：The porcine cornea descemet's membrane prepared, sucks antibiotic liquid, to pig after bullet
In power layer surface add 1-10 μ g/ml Laminin lens (Laminin, LN, are purchased from Biolamina companies, article No. LN511,
LN521) or FNC Coating Mix (being purchased from AthenaES companies, article No. 0470) are coated with to promote cell adhesion, coating
Remove coating liquid after enough time, air-dry standby.By the inducing cell digested by 150-2000 cell/mm2Density connects
Boar entocornea, be placed in 37 DEG C, 5%CO2 incubators it is adherent overnight after, change the left side of fresh differential medium culture one week
The right side, changes liquid daily；
The 1-10 μ g/ml Laminin lens refer to the concentration after being diluted using 1 × DPBS, fresh are matched somebody with somebody using preceding every time
System, the coating time is to be stayed overnight within 1 hour or 4 DEG C in 37 DEG C；The FNC Coating Mix are commodity instant, and the coating time is
Before inoculating cell, room temperature is coated with 1 minute or so.
Another object of the present invention is to provide the organizational project that the construction method of above-mentioned tissue engineering comea endothelium is obtained
Corneal endothelium.
A further object of the present invention is that the tissue engineering comea endothelium that the above-mentioned construction method of offer is obtained is (interior in cornea
Skin) transplanting in purposes.
As shown in subsequent embodiment of the present invention, the construction method and its organizational project of tissue engineering comea endothelium of the invention
Corneal endothelium, at least has the advantage that：Compared to amnion or other carriers, descemet's membrane used herein is cornea in itself
Normal configuration, will not be excessively soft, transparent good.Used seed cell comes for induced differentiation of embryonic stem cells, not by
The low limitation of primary cell multiplication capacity.Also, tissue engineering comea endothelium preparation process of the present invention is simple, the wide hair in source, easily
In operation.
Brief description of the drawings
Fig. 1 is tissue engineering comea endothelium Alizarin red staining figure constructed in one embodiment of the invention；
Fig. 2 is tissue engineering comea endothelium expression endothelial cell important symbol Na constructed in one embodiment of the invention+-K+- ATPase, ZO-1 scheme.
Embodiment
Further technical scheme is illustrated below by way of specific embodiment, it should be understood that be only this hair below
Bright exemplary illustration, is not intended to limit the invention scope of the claims.
Embodiment 1
1. prepare porcine cornea descemet's membrane：Take fresh complete porcine cornea, using containing blue or green chain it is dual anti-(blue or green chain it is dual anti-(100 ×,
Be purchased from Corning companies of the U.S.)) sterile saline immersion, rinse 20 minutes after, endothelium is placed in Stereo microscope up
Under, removal porcine cornea endothelial cell is carefully wiped using cotton swab, porcine cornea is fixed using pincers, remnants iris root is first torn off
Portion and exposure descemet's membrane free fraction, then carefully grip the free place of pig descemet's membrane using tack tweezer, light to Central corneal
Light tear makes pig descemet's membrane completely be separated with corneal stroma.Note operating force, it is to avoid descemet's membrane is torn.
By the porcine cornea descemet's membrane completely separated it is smooth be enclosed within internal diameter be 9.0mm antiseptic rubber annulus on, interior surface
It is attached in 35mm culture plates center, super-clean bench and dries upwards, ultraviolet irradiation sterilizes overnight, is soaked using preceding addition antibiotic
It is placed in 37 DEG C, 5%CO2Incubator is standby.
2. induce human pluripotent stem cells：After passage cellar culture human pluripotent stem cells (strain of H1 human embryo stem cells, from
ATCC is obtained) culture to suitable size is to start induction, uses induction containing RA (being purchased from Sigma companies, article No. R2625) to train
Support base continuous induction 5 days, visible human pluripotent stem cells clone expands under mirror, adjacent clone is merged, and clones inner cell volume
Become big, cellular morphology and polygon is changed into from circular or oval, arrangement is close, clone's periphery cell is in fusiformis threadiness.Terminate
Induction, removes inducing culture, adds 2ml without Ca2+、Mg2+PBS wash 2 times, add 0.25% pancreatin -0.02%
EDTA solution 1.5ml (Trypsin-EDTA solution, 10x are purchased from Sigma companies, article No. T4174), removes threadiness thin
After born of the same parents and pancreatin, remaining cell clone places incubator and continues to digest, using containing ROCK inhibitor when most cell detachment
5%DKSFM differential mediums (being purchased from Invitrogen companies, article No. 10744019) are terminated, resuspended, piping and druming to unicellular rear meter
Number is standby.
The human pluripotent stem cells clone of the suitable size refers to；About 60-100 cell under inverted phase contrast microscope/gram
It is grand, clone uniform in size, medium density.
The inducing culture containing RA is to add following material system on knockout DMEM/F12 basal mediums
Into：Beta -mercaptoethanol, glutamine, bFGF, NEAA, KSR, RA.After addition, beta -mercaptoethanol concentration is 0.1mM, glutamine
Concentration is that 0.1mM, bFGF concentration are that 4ng/ml, NEAA are 20% (volume fraction) that 0.1mM, KSR concentration are total amount of liquid, RA
Concentration is 1 μM.
The 5%DKSFM differential mediums containing ROCK inhibitor be in DKSFM basal mediums add FBS and
What ROCK inhibitor was made.Its concentration be respectively 5% (volume fraction) of total amount of liquid, 10 μM.
3. build engineered corneal endothelium：The porcine cornea descemet's membrane prepared is taken out, antibiotic liquid is sucked, to
Surface adds FNC Coating Mix and is coated with to promote cell adhesion in porcine cornea descemet's membrane, and room temperature is coated with about 1 minute
After remove coating liquid, be placed in Biohazard Safety Equipment air-dry it is standby.
By the inducing cell digested by 150-2000 cell/mm2Density Pigs Inoculated entocornea, is placed in 37
DEG C, 5%CO2Incubator is adherent overnight, changes within second day fresh differential medium culture one week or so, visible descemet's membrane under mirror
It is clear that the endothelial cell in upper human pluripotent stem cells source arranges close, form rule, cell boundaries.
The FNC Coating Mix are commodity instant, and the coating time is before inoculating cell, room temperature is coated with 1 minute left side
It is right.
As shown in figure 1, obtaining observation of cell under tissue engineering comea endothelium, phase contrast microscope using method in embodiment 1
Cover with whole descemet's membrane.Skin graft in the tissue engineering comea of structure is dyed 5 minutes using alizarin red dye liquor, physiology salt is dipped in
Carefully rinsed in water, be placed in clean slide light Microscopic observation.Wherein, a：Visible constructed tissue engineering comea endothelium is thin under mirror
Born of the same parents' arrangement is close, cell boundaries are clear；b；Constructed tissue engineering comea endothelium Alizarin red staining, it is seen that cell boundaries are coloured
Clearly, form rule, activity is good, and karyon nowhere contaminates.As a result illustrate：The corneal endothelium that the inventive method is obtained has good
Form and cytoactive.
As shown in Fig. 2 obtaining tissue engineering comea endothelium using method in embodiment 1, after Microscopic observation cell is covered with, make
With skin graft in 4% paraformaldehyde immobilizing corneal, PBS washing adds Na+-K+The corresponding primary antibody of-ATPase, ZO-1, secondary antibody
After incubation, fluorescence microscopy Microscopic observation.Wherein, a：Tissue engineering comea endothelium expression endothelial cell liquid pump function mark Na+-K+-ATPase；b：The close albumen ZO-1 of tissue engineering comea endothelium expression endothelial cell structure mark.As a result illustrate：This
The tissue engineering comea endothelium that inventive method is obtained expresses the important 26S Proteasome Structure and Function mark of corneal endothelium, is exercised in cornea
The basis of skin corresponding function.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of construction method of tissue engineering comea endothelium, comprises the following steps：
1) porcine cornea descemet's membrane is prepared：Porcine cornea is removed into porcine cornea endothelial cell, makes pig descemet's membrane and corneal stroma complete
Whole separation, by the porcine cornea descemet's membrane completely separated dry, sterilize it is standby；
2) cultivate, induce human pluripotent stem cells：By the human pluripotent stem cells culture of cellar culture to suitable size, trained using induction
Base induction is supported, become big, cellular morphology to human pluripotent stem cells clone's inner cell volume is changed into polygon from circular or oval, arranges
Row are close, and clone's periphery cell terminates induction in fusiformis threadiness；It will make after the human pluripotent stem cells Clone Digestion for terminating induction
It is resuspended and count standby with differential medium；
3) tissue engineering comea endothelium is built：By step 1) obtain porcine cornea descemet's membrane, add coating buffer be coated with, then
By step 2) the induction human pluripotent stem cells that obtain are inoculated in step 1) the porcine cornea descemet's membrane that obtains, after cell attachment, more
Renew after fresh differential medium culture, obtain tissue engineering comea endothelium.
2. the construction method of tissue engineering comea endothelium according to claim 1, it is characterised in that the step 1) use
Porcine cornea plant the complete total corneal of piece diametric requirements to meet to build or possess the porcine cornea of descemet's membrane.
3. the construction method of tissue engineering comea endothelium according to claim 1, it is characterised in that the step 1) in institute
Dry sterilization is stated to be processed as using x ray irradiation x or add antibiotic medicine sterilization treatment.
4. the construction method of tissue engineering comea endothelium according to claim 1, it is characterised in that the step 2) in institute
The size for stating the human pluripotent stem cells of suitable size is 60-100 cell/clone.
5. the construction method of tissue engineering comea endothelium according to claim 4, it is characterised in that：The inducing culture
It is made by adding following material on DMEM/F12 basal mediums：Beta -mercaptoethanol, glutamine, bFGF, NEAA, KSR,
RA；Preferably, the beta -mercaptoethanol concentration be 0.1mM, glutamine concentration be 0.1mM, bFGF concentration be 4-8ng/ml,
NEAA be 20% (volume fraction) that 0.1mM, KSR concentration are total amount of liquid, RA concentration be 0.5-2 μM.
6. the construction method of tissue engineering comea endothelium according to claim 4, it is characterised in that：The differential medium
For the 5%DKSFM culture mediums containing ROCK inhibitor.
7. the construction method of tissue engineering comea endothelium according to claim 1, it is characterised in that the step 3) in institute
It is to be adapted to the extracellular matrix that corneal endothelium is attached to state coating buffer, such as Laminin lens, FNC or Matrigel.
8. the construction method of the tissue engineering comea endothelium according to claim 1~7 any one, it is characterised in that bag
Include following steps：
1) porcine cornea descemet's membrane is prepared：The porcine cornea that fresh or glycerine is preserved, is soaked using the sterile saline containing antibiotic
Bubble, rinse 20 minutes after, endothelium is placed under Stereo microscope up, using cotton swab carefully wipe removal porcine cornea endothelium it is thin
Born of the same parents, porcine cornea is fixed using pincers, remnants iris root and exposure descemet's membrane free fraction is first torn off, then using flat
Head tweezer carefully grips the free place of pig descemet's membrane, and gently being torn to Central corneal makes pig descemet's membrane completely divide with corneal stroma
From.Operation should be noted dynamics, once descemet's membrane rupture curling can not be used.
It is enclosed within the porcine cornea descemet's membrane completely separated is smooth on internal diameter≤11.0mm antiseptic rubber annulus, endothelium is upwardly
Be attached at culture plate be allowed to dry, sterilizing, using it is preceding addition antibiotic immersion be placed in 37 DEG C, 5%CO2 incubators it is standby；
2) human pluripotent stem cells are induced：The human pluripotent stem cells culture of cellar culture to suitable size is to begin to use containing RA's
Visible human pluripotent stem cells clone expands and connected under inducing culture continuous induction 3-5 days, mirror, and clone's inner cell volume becomes
Greatly, cellular morphology is changed into polygon from circular or oval, and arrangement is close, and clone's periphery cell is in fusiformis threadiness.It will terminate
Use the 5%DKSFM differential mediums containing ROCK inhibitor resuspended after the human pluripotent stem cells Clone Digestion of induction and count standby
With；
The human pluripotent stem cells clone of the suitable size refers to：About 60-100 cell/clone under inverted phase contrast microscope, gram
It is grand uniform in size, medium density；
The inducing culture containing RA is made of to add following material on knockout DMEM/F12 basal mediums：
Beta -mercaptoethanol, glutamine, bFGF, NEAA, KSR, RA；After addition, beta -mercaptoethanol concentration is 0.1mM, glutamine is dense
Degree is that 0.1mM, bFGF concentration are that 4-8ng/ml, NEAA are 20% (volume fraction) that 0.1mM, KSR concentration are total amount of liquid, RA
Concentration is 0.5-2 μM；
The 5%DKSFM differential mediums containing ROCK inhibitor are that FBS and ROCK suppressions are added in DKSFM basal mediums
What preparation was made.Its concentration be respectively 5% (volume fraction) of total amount of liquid, 5-20 μM；
3) tissue engineering comea endothelium is built：The porcine cornea descemet's membrane prepared, sucks antibiotic liquid, to pig descemet's membrane
Interior surface adds 1-10 μ g/ml Laminin lens or compound coating buffer is coated with to promote cell adhesion, is coated with enough time
After remove coating liquid, air-dry it is standby.By the inducing cell digested by 150-2000 cell/mm2Density is inoculated with porcine cornea
Descemet's membrane, be placed in 37 DEG C, 5%CO2 incubators it is adherent overnight after, change fresh differential medium culture one week or so, daily
Change liquid；
The Laminin lens are the concentration after being diluted using 1 × DPBS, every time using preceding Fresh, and the coating time is in 37
DEG C 1 hour or 4 DEG C overnight；
The compound coating buffer is commodity instant, and the coating time is before inoculating cell, room temperature is coated with 1 minute or so.
9. the organizational project that a kind of construction method of tissue engineering comea endothelium as described in any one of claim 1~8 is obtained
Corneal endothelium.
10. purposes of the tissue engineering comea endothelium according to claim 9 in cornea or corneal endothelium transplanting.
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