CN108578778A - A kind of cell for Ocular surface damage reparation plants piece and preparation method thereof - Google Patents
A kind of cell for Ocular surface damage reparation plants piece and preparation method thereof Download PDFInfo
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- CN108578778A CN108578778A CN201810473890.6A CN201810473890A CN108578778A CN 108578778 A CN108578778 A CN 108578778A CN 201810473890 A CN201810473890 A CN 201810473890A CN 108578778 A CN108578778 A CN 108578778A
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- cells
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Classifications
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- Dispersion Chemistry (AREA)
- Urology & Nephrology (AREA)
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Abstract
The invention discloses a kind of cells for Ocular surface damage reparation to plant piece, including cell membrane lamella and fibrin layer double-layer structure, the cell membrane lamella is the membrane structure that the thickness being arranged to make up by 5~7 layers of epithelial cell is 50 μm~150 μm, the fibrin layer be 200 μm~500 μm by the thickness that Fibrin Glue is formed loose porous membrane structure；The invention also discloses the preparation methods that a kind of cell for Ocular surface damage reparation plants piece, including step：One, the preparation of cell membrane lamella；Step 2: the preparation of fibrin layer.The present invention is conducive to the reparation and regeneration of corneal epithelium, reduces operating difficulty, improves cell utilization rate and cell plants piece preparation efficiency, improve cell and plant piece survival rate, highly practical, using effect is good, convenient for promoting the use of.
Description
Technical field
The invention belongs to tissue engineering technique fields, and in particular to a kind of cell for Ocular surface damage reparation plant piece and its
Preparation method.
Background technology
Cornea can be divided into five layers from outside to inside, be corneal epithelium, anterior limiting lamina respectively（Also known as Bowman layers, preceding elastic force
Layer）, corneal stroma, posterior limiting lamina（Also known as Descemet layers, descemet's membrane）And corneal endothelial layer.Wherein, corneal epithelium thickness
It 50~100 μm, is made of 5~6 layers of nonkeratinizing pavement epithelium cells.Cell arrangement is neat, is easily detached with bowman's lamina.
Keep the complete of corneal epithelium and without vascularization state for maintaining the physiological function of cornea particularly significant.Theoretically, compare
Slight Ocular surface damage can pass through limbal stem cell（Corneal limbal stem cell, CLSC）Proliferation, orientation
Migrate and break up the reparation for completing corneal epithelial layer.However, many factors clinically, including mechanical damage, chemical burn,
High temperature scald, infection, ulcer and other disease of cornea, usually can all cause the missing of limbal stem cell, cause damaged corneal
Epithelial layer can not be repaired, so there is the opacity of the cornea, new vessels are grown into, influence twenty-twenty vision, can cause to blind when serious.
Treatment eye surface diseases most efficient method is to carry out corneal transplantation.But corneal allograft is depended on and is contributed, by
To the very big limitation of donor source, a large amount of clinical needs are cannot be satisfied.And self limbal transplantation is also by source deficiency
Limitation, while the safety of strong eye also being made to be on the hazard.Therefore, the treatment of Ocular surface damage is still more intractable at present asks
Topic.
Organizational engineering and biomaterial the reach of science provide many new idea and methods to repair Ocular surface damage.
It attempts to be at most to use amnion（Or other biological film）Covering is damaged ocular, utilizes the collagen, transparent contained in amnion
Matter acid and growth factor isoreactivity ingredient, reduce inflammation reaction, inhibits scar and vascularization, to protection and repairs impaired eye
Table epithelial layer.Further way is to use amnion as basilar memebrane, by its area load cell, being built into a group weaver
Journey corneal epithelium plants piece for treating Ocular surface damage.Application No. is the Chinese patents of 200410019341.X to disclose one kind
" cornea edge stem cell tissue engineering composite body and preparation method thereof, it uses de- cell amnion as carrier, and corneal limbus is dry thin
Born of the same parents' inoculation is formed on its surface complex for treating Ocular surface damage.Application No. is 200410083822.7 Chinese patents to disclose
A kind of medical cornea paster and preparation method, it is will be on the mescenchymal stem cell of the autologous amnion that is seeded in that treated
It forms compound and is used for Ocular surface healing.Application No. is 201110400135.3 Chinese patents to disclose a kind of culture people's corneal limbus
The method that stem cell plants piece is that limbal stem cell is seeded on the amnion preserved through the freeze-drying that cobalt -60 sterilizes, is formed
Cell plants piece.The above invention is all using amnion as cell carrier, and there is problems with：（1）Through de- cell, drying, sterilizing
And etc. processing amnion, bioactive ingredients therein are lost in or destroy, it is difficult to play expected effect；（2）Point of amnion
Sub- permeability is poor, and the cell after transplanting on amnion is difficult to obtain nutrition and oxygen, easily leads to cell death；（3）Amnion carrying
Compact structure, degradation speed are slow, influence the integration of cell and ocular；（4）To amnion surface seeding cell difficulty, big, cell is lost in
Rate is high, and cell is difficult to form cladding, differs greatly with natural cornea epithelial layer structure.
Application No. is 200480003698.7 Chinese patent disclose a kind of cell sheet being used to form corneal epithelium and
Its manufacturing method and application method, it is will to form the cell inoculation of corneal epithelium in the culture of temperature-responsive polymer peridium
On ware, as needed by after cell multiple stratification, polymeric hydrophilic performance is controlled by temperature, achievees the purpose that detach cell sheet,
The cell sheet does carrier without amnion or other biological film.However, the cell sheet poor toughness of this method structure, from culture dish bottom point
From when there is still a need for being supported by pvdf membrane, cannot be satisfied clinically complicated operation requirement.
Invention content
In view of the above-mentioned deficiencies in the prior art, the technical problem to be solved by the present invention is that providing a kind of for ocular
The cell of injury repair plants piece, and cell membrane lamella remains the growth factor isoreactivity ingredient generated in cell cultivation process,
Be conducive to the reparation and regeneration of corneal epithelium；Its fibrin layer has good mechanical performance, can play maintenance cell
The effect of membrane layer form also provides mechanics for cell membrane lamella and supports, cell membrane lamella is enable to bear more complicated operation
Operation reduces operating difficulty；Its fibrin layer can ensure the nutrition supply of cell, improve cell and plant piece survival rate, and dredge
The structure of pine makes basal layer after completing its mechanics supporting function, cell membrane lamella can be allow directly to participate in fast degradation
Ocular epithelial layer reparation.
In order to solve the above technical problems, the technical solution adopted by the present invention is：A kind of cell for Ocular surface damage reparation
Plant piece, it is characterised in that：Including cell membrane lamella and fibrin layer double-layer structure, the cell membrane lamella is by 5~7 layers
The membrane structure that the thickness that cells line is constituted is 50 μm~150 μm, the fibrin layer is to be formed by Fibrin Glue
The loose porous membrane structure that thickness is 200 μm~500 μm.
A kind of above-mentioned cell for Ocular surface damage reparation plants piece and preparation method thereof, it is characterised in that：The epithelium
Cell is corneal epithelial cell, Buccal mucosa cell, amniotic epithelial cells and thin by the epithelium of Derived from Mesenchymal Stem Cells
One or more mixtures in born of the same parents.
The invention also discloses a kind of method and step is simple, avoid that cell is lost in, repeatedly inoculation and cellular layer are formed not
Uniform problem improves the system of the cell plant piece for Ocular surface damage reparation of cell utilization rate and cell plant piece preparation efficiency
Preparation Method, which is characterized in that this approach includes the following steps：
Step 1: the preparation of cell membrane lamella, detailed process are：
Epithelial cell is seeded in progress cell patch culture in the cells Transwell by step 101；It is cultivated using DMEM high sugar
Base simultaneously changes once for every two days, and after cultivating 15~20 days, cell patch is formed；
Step 102, the liquid level for reducing DMEM high glucose mediums carry out air-liquid interface culture, change daily to cell patch layer height
DMEM high glucose medium, after 7 days, inverted microscope observation forms continuous epithelium layer, cell membrane lamella culture
Terminate；
Step 2: the preparation of fibrin layer, detailed process are：
After DMEM high glucose mediums in step 201, removal Transwell cells, then remaining DMEM washed away using cleaning solution
High glucose medium；
Step 202 uses the CaCl that substance withdrawl syndrome is 40mmol/L2Solution compound concentration is 20U/ml~100U/ml's
Thrombin solution；
Step 203 prepares the fibrin original solution that quality-volumetric concentration is 0.02g/ml~0.1g/ml using pure water；
Step 204 takes thrombin solution 0.5ml in step 202, the fibrin original solution 0.5ml in step 203 to adopt
Thrombin solution and fibrin original solution are uniformly applied on cell membrane lamella simultaneously with double copmled medicine mixer, waiting 2min~
After 5min, Fibrin Glue solidification, fibrin layer preparation terminates；
Step 3: the fibrin layer prepared is slowly taken off from the bottom surface of the cells Transwell simultaneously with cell membrane lamella
Under, it has been made and has planted piece for the cell of Ocular surface damage reparation.
Above-mentioned method, it is characterised in that：A diameter of 12mm of Transwell cells described in step 101 is thin by epithelium
It is 2 × 10 that born of the same parents, which are seeded in the inoculum density in the cells Transwell,4A/cm2~1 × 105A/cm2。
Above-mentioned method, it is characterised in that：It carries out being in temperature when cell patch culture in step 101 being 37 DEG C, quality
The CO that percent concentration is 5%2It is carried out under atmospheric condition.
Above-mentioned method, it is characterised in that：Contain mass percent concentration in DMEM high glucose mediums described in step 101
For 10% fetal calf serum, quality-volumetric concentration be 30mg/ml~70mg/ml vitamin C, quality-volumetric concentration be 2ng/
The epidermal growth factor of ml~5ng/ml, the bovine brain pituitary extract and matter that quality-volumetric concentration is 10 μ of μ g/ml~30 g/ml
Amount-volumetric concentration is the sodium selenite of 2ng/ml~5ng/ml.
Above-mentioned method, it is characterised in that：Cleaning solution described in step 201 is phosphate buffer.
Compared with the prior art, the present invention has the following advantages：
1, it includes cell membrane lamella and fibrin layer double-layer structure that cell of the invention, which plants piece, and cell membrane lamella has and cornea
The similar institutional framework of epithelium layer height, and cell membrane lamella remains growth factor generated in cell cultivation process etc. and lives
Property ingredient, is conducive to the reparation and regeneration of corneal epithelium.
2, when cell of the invention plants piece preparation, it is small only need to epithelial cell high density to be disposably seeded in Transwell
It can be formed in room, without to biomembrane material surface seeding cell, avoiding, cell is lost in, repeatedly inoculation and cellular layer are formed
Non-uniform problem, improves cell utilization rate and cell plants piece preparation efficiency.
3, the present invention has good mechanical performance by the fibrin layer that Fibrin Glue is formed, and it is thin can to play maintenance
The effect of after birth platelet morphology also provides mechanics for cell membrane lamella and supports, cell membrane lamella is enable to bear more complicated hand
Art operates, and reduces operating difficulty.
4, the present invention by the fibrin layer that Fibrin Glue is formed there is loose internal structure, this structure to allow to seek
It supports substance and oxygen passes through, can ensure the nutrition supply of cell, improve cell and plant piece survival rate；In addition, loose structure makes
Basal layer can allow cell membrane lamella directly to participate in ocular epithelial layer after completing its mechanics supporting function with fast degradation
It repairs.
5, the present invention's is highly practical, and using effect is good, convenient for promoting the use of.
Below by drawings and examples, technical scheme of the present invention will be described in further detail.
Description of the drawings
Fig. 1 is the structural schematic diagram that the present invention plants piece for the cell of Ocular surface damage reparation.
Fig. 2 is the method flow block diagram for the preparation method that the present invention plants piece for the cell of Ocular surface damage reparation.
Fig. 3 A are that the cell prepared using the present invention plants the animal corneal that piece prepared when eye epidermis injury reparative experiment
Limbal stem cell lacks eye surface diseases illustraton of model.
Fig. 3 B are the figure after Ocular surface healing when planting piece progress eye epidermis injury reparative experiment using cell prepared by the present invention.
Reference sign:
1-cell membrane lamella；2-fibrin layers.
Specific implementation mode
The cell for Ocular surface damage reparation of the present invention plants piece, is described by 1~embodiment of embodiment 4；The present invention
For Ocular surface damage reparation cell plant piece preparation method, described by 5~embodiment of embodiment 7.
Embodiment 1
As shown in Figure 1, the cell for Ocular surface damage reparation of the present embodiment plants piece, including cell membrane lamella 1 and fibrin
2 double-layer structure of layer, the cell membrane lamella 1 is the film that the thickness being arranged to make up by 5~7 layers of epithelial cell is 50 μm~150 μm
Structure, the fibrin layer 2 be 200 μm~500 μm by the thickness that Fibrin Glue is formed loose porous membrane structure.
The cell membrane lamella 1 similar to natural cornea epithelium multilayer structure, the fibrin layer 2 have certain tensile strength,
Bonding force after the fibrin layer 2 is solidified by fibrin original solution is fitted closely with cell membrane lamella 1, to maintain
The form of cell membrane lamella 1, and increase the mechanical strength of cell membrane lamella 1.
In the present embodiment, the epithelial cell be corneal epithelial cell, Buccal mucosa cell, amniotic epithelial cells and
By the epithelial cell of Derived from Mesenchymal Stem Cells.
Embodiment 2
The present embodiment is as different from Example 1：The epithelial cell is corneal epithelial cell, Buccal mucosa cell, sheep
Film epithelial cell and by arbitrary two kinds of the mixture in the epithelial cell of Derived from Mesenchymal Stem Cells.Remaining structure with implementation
Example 1 is identical.
Embodiment 3
The present embodiment is as different from Example 1：The epithelial cell is corneal epithelial cell, Buccal mucosa cell, sheep
Film epithelial cell and by arbitrary three kinds of the mixture in the epithelial cell of Derived from Mesenchymal Stem Cells.Remaining structure with implementation
Example 1 is identical.
Embodiment 4
The present embodiment is as different from Example 1：The epithelial cell is corneal epithelial cell, Buccal mucosa cell, sheep
The mixture of film epithelial cell and epithelial cell by Derived from Mesenchymal Stem Cells.Remaining structure is same as Example 1.
Embodiment 5
As shown in Fig. 2, the cell for Ocular surface damage reparation of the present embodiment plants the preparation method of piece, include the following steps：
Step 1: the preparation of cell membrane lamella 1, detailed process are：
New zealand white rabbit corneal epithelial cell is seeded in progress cell patch culture in the cells Transwell by step 101；
It using DMEM high glucose mediums and changes within every two days once, after cultivating 18 days, cell patch is formed；
In the present embodiment, epithelial cell is seeded in the cells Transwell by a diameter of 12mm of the cells Transwell
Inoculum density be 5 × 104A/cm2。
In the present embodiment, carry out cell patch culture when be in the CO that temperature is 37 DEG C, mass percent concentration is 5%2Gas
It is carried out under the conditions of atmosphere.
In the present embodiment, the fetal calf serum for being 10% containing mass percent concentration in the DMEM high glucose mediums
（FBS）, quality-volumetric concentration be 50mg/ml vitamin C, quality-volumetric concentration be 3.5ng/ml epidermal growth factor,
The bovine brain pituitary extract that quality-volumetric concentration is 20 μ g/ml and the sodium selenite that quality-volumetric concentration is 3.5ng/ml.
Step 102, the liquid level for reducing DMEM high glucose mediums carry out air-liquid interface culture to 1 height of cell membrane lamella,
A DMEM high glucose medium is changed daily, after 7 days, inverted microscope observation forms continuous epithelium layer, cell patch
1 culture of layer terminates；Continuous epithelium layer can be formed using air-liquid interface culture, connected between cell stronger.
Step 2: the preparation of fibrin layer 2, detailed process are：
After DMEM high glucose mediums in step 201, removal Transwell cells, then remaining DMEM washed away using cleaning solution
High glucose medium；
In the present embodiment, the cleaning solution is phosphate buffer.
Step 202 uses the CaCl that substance withdrawl syndrome is 40mmol/L2Solution compound concentration is the fibrin ferment of 50U/ml
Solution；
Step 203 prepares the fibrin original solution that quality-volumetric concentration is 0.1g/ml using pure water；
Step 204 takes thrombin solution 0.5ml in step 202, the fibrin original solution 0.5ml in step 203 to adopt
Thrombin solution and fibrin original solution are uniformly applied on cell membrane lamella 1 simultaneously with double copmled medicine mixer, wait for 2min
Afterwards, Fibrin Glue solidifies, and the preparation of fibrin layer 2 terminates；
Step 3: the fibrin layer 2 prepared and cell membrane lamella 1 is slow from the bottom surface of the cells Transwell simultaneously
It takes off, has been made and has planted piece for the cell of Ocular surface damage reparation.
In the present embodiment, the process that is separately cultured of the new zealand white rabbit corneal epithelial cell used in step 101 is：
First, after new zealand white rabbit being anaesthetized, 1 ~ 3mm corneal limbal tissues are obtained, using containing antibiotic under gnotobasis
Phosphate buffer clean 3 times；The tissue is placed in 15ml centrifuge tubes again, uses the Dispase II of a concentration of 1.2U/ml
It is digested 4 ~ 8 hours at a temperature of 4 DEG C, takes out tissue careful separation and go out corneal epithelium, it is dense which is placed on quality percentage
Degree terminates cleaning, centrifugation after digestion, obtains on cornea to digest 10min at a temperature of 37 DEG C in 0.25% trypsin solution
Chrotoplast；Then, by corneal epithelial cell in the CO that temperature is 37 DEG C, mass percent concentration is 5%2It is carried out under atmospheric condition
Amplification cultivation, used medium are no calcium ions and magnesium ions DMEM/F-12（1:1）Culture medium, wherein containing 5ng/ml epidermal growth factors
Son, 20 μ g/ml bovine brain pituitary extracts, 15 μ g/ml pigment epithelium growth factors；Epithelial cell was expanded to for the 3rd generation.
Embodiment 6
As shown in Fig. 2, the cell for Ocular surface damage reparation of the present embodiment plants the preparation method of piece, include the following steps：
Step 1: the preparation of cell membrane lamella 1, detailed process are：
New zealand white rabbit Buccal mucosa cell is seeded in progress cell patch training in the cells Transwell by step 101
It supports；It using DMEM high glucose mediums and changes within every two days once, after cultivating 20 days, cell patch is formed；
In the present embodiment, epithelial cell is seeded in the cells Transwell by a diameter of 12mm of the cells Transwell
Inoculum density be 1 × 105A/cm2。
In the present embodiment, carry out cell patch culture when be in the CO that temperature is 37 DEG C, mass percent concentration is 5%2Gas
It is carried out under the conditions of atmosphere.
In the present embodiment, the fetal calf serum for being 10% containing mass percent concentration in the DMEM high glucose mediums
（FBS）, quality-volumetric concentration be 70mg/ml vitamin C, quality-volumetric concentration be 5ng/ml epidermal growth factor, matter
The bovine brain pituitary extract that amount-volumetric concentration is 30 μ g/ml and the sodium selenite that quality-volumetric concentration is 5ng/ml.
Step 102, the liquid level for reducing DMEM high glucose mediums carry out air-liquid interface culture to 1 height of cell membrane lamella,
A DMEM high glucose medium is changed daily, after 7 days, inverted microscope observation forms continuous epithelium layer, cell patch
1 culture of layer terminates；Cell membrane lamella can be made to realize epithelialization using air-liquid interface culture, form similar natural cornea surface layer
Structure.
Step 2: the preparation of fibrin layer 2, detailed process are：
After DMEM high glucose mediums in step 201, removal Transwell cells, then remaining DMEM washed away using cleaning solution
High glucose medium；
In the present embodiment, the cleaning solution is phosphate buffer.
Step 202 uses the CaCl that substance withdrawl syndrome is 40mmol/L2Solution compound concentration is the fibrin ferment of 20U/ml
Solution；
Step 203 prepares the fibrin original solution that quality-volumetric concentration is 0.05g/ml using pure water；
Step 204 takes thrombin solution 0.5ml in step 202, the fibrin original solution 0.5ml in step 203 to adopt
Thrombin solution and fibrin original solution are uniformly applied on cell membrane lamella 1 simultaneously with double copmled medicine mixer, waited for
After 3.5min, Fibrin Glue solidification, the preparation of fibrin layer 2 terminates；
Step 3: the fibrin layer 2 prepared and cell membrane lamella 1 is slow from the bottom surface of the cells Transwell simultaneously
It takes off, has been made and has planted piece for the cell of Ocular surface damage reparation.
In the present embodiment, the new zealand white rabbit Buccal mucosa cell that is used in step 101 is separately cultured process
For：
First, after drawing piperazine and 40mg/kg ketalars to anaesthetize new zealand white rabbit using 4mg/kg hydrochloric acid plugs, 3mm is obtained
× 3mm mucous membrane of mouth is cleaned 3 times under gnotobasis using the phosphate buffer containing antibiotic；It reuses a concentration of
The Dispase II of 1.5U/ml digests 4 hours at a temperature of 4 DEG C, the trypsase that use quality percentage concentration is 0.25% later
20min is digested in solution at room temperature, cleaning, centrifugation after digestion is terminated, obtains Buccal mucosa cell；Then, by oral cavity
Mucosal epithelial cells are in the CO that temperature is 37 DEG C, mass percent concentration is 5%2Amplification cultivation, training used are carried out under atmospheric condition
It is no calcium ions and magnesium ions DMEM/F-12 to support base（1:1）Culture medium, wherein containing mass percentage concentration be 0.4% dimethyl sulfoxide (DMSO),
Epidermal growth factor that quality-volumetric concentration is 5ng/ml, quality-volumetric concentration are that the insulin of 4 μ g/ml, quality-volume are dense
Spend the hydrocortisone for being 1ng/ml, quality-volumetric concentration is the transferrins of 5 μ g/ml, quality-volumetric concentration is 5ng/ml
Sodium selenite, the bovine brain pituitary extract that quality-volumetric concentration is 30 μ g/ml and color that quality-volumetric concentration is 10 μ g/ml
Plain epidermal derived factors；Epithelial cell was expanded to for the 3rd generation.
Embodiment 7
As shown in Fig. 2, the cell for Ocular surface damage reparation of the present embodiment plants the preparation method of piece, include the following steps：
Step 1: the preparation of cell membrane lamella 1, detailed process are：
Step 101 will be seeded in the cells Transwell by the epithelial cell of mesenchymal stem cells differentiation and carry out cell membrane
Piece culture；It using DMEM high glucose mediums and changes within every two days once, after cultivating 15 days, cell patch is formed；
In the present embodiment, epithelial cell is seeded in the cells Transwell by a diameter of 12mm of the cells Transwell
Inoculum density be 2 × 104A/cm2。
In the present embodiment, carry out cell patch culture when be in the CO that temperature is 37 DEG C, mass percent concentration is 5%2Gas
It is carried out under the conditions of atmosphere.
In the present embodiment, the fetal calf serum for being 10% containing mass percent concentration in the DMEM high glucose mediums
（FBS）, quality-volumetric concentration be 30mg/ml vitamin C, quality-volumetric concentration be 2ng/ml epidermal growth factor, matter
The bovine brain pituitary extract that amount-volumetric concentration is 10 μ g/ml and the sodium selenite that quality-volumetric concentration is 2ng/ml.
Step 102, the liquid level for reducing DMEM high glucose mediums carry out air-liquid interface culture to 1 height of cell membrane lamella,
A DMEM high glucose medium is changed daily, after 7 days, inverted microscope observation forms continuous epithelium layer, cell patch
1 culture of layer terminates；Continuous epithelium layer can be formed using air-liquid interface culture.
Step 2: the preparation of fibrin layer 2, detailed process are：
After DMEM high glucose mediums in step 201, removal Transwell cells, then remaining DMEM washed away using cleaning solution
High glucose medium；
In the present embodiment, the cleaning solution is phosphate buffer.
Step 202 uses the CaCl that substance withdrawl syndrome is 40mmol/L2Solution compound concentration is the blood coagulation of 100U/ml
Enzyme solutions；
Step 203 prepares the fibrin original solution that quality-volumetric concentration is 0.02g/ml using pure water；
Step 204 takes thrombin solution 0.5ml in step 202, the fibrin original solution 0.5ml in step 203 to adopt
Thrombin solution and fibrin original solution are uniformly applied on cell membrane lamella 1 simultaneously with double copmled medicine mixer, wait for 5min
Afterwards, Fibrin Glue solidifies, and the preparation of fibrin layer 2 terminates；
Step 3: the fibrin layer 2 prepared and cell membrane lamella 1 is slow from the bottom surface of the cells Transwell simultaneously
It takes off, has been made and has planted piece for the cell of Ocular surface damage reparation.
In the present embodiment, the epithelial cell by mesenchymal stem cells differentiation that is used in step 101 is separately cultured
And atomization is：
First, after 4 ~ 5 monthly age new zealand white rabbits being anaesthetized, from ilium bone marrow extraction 5ml, Ficoll human lymphocytes point are used
Chaotropic separating bone marrow mesenchymal stem, be added containing mass percentage concentration be 10% fetal calf serum DMEM culture mediums 3ml ~
4ml blows even and fine born of the same parents, is inoculated in culture bottle, under the CO2 atmospheric conditions that temperature is 37 DEG C, mass percent concentration is 5% into
Row incubator culture, when cell growth to 80% or more floor space, the trypsase for being 0.25% by cell mass percentage concentration
It is digested in solution, is dispersed into unicellular, be inoculated in new culture bottle, pass on for the first time, rule changes liquid, passage one in 4 ~ 6 days
It is secondary, until the third generation；Then, third generation mesenchymal stem cell is collected, is inoculated in 10cm culture dishes, is added with 1 × 105 quantity
Enter epithelial cell induction liquid, using DMEM low sugar culture mediums, wherein being given birth to containing 10% fetal calf serum, 10ng/ml cuticulated epitheliums
After cultivating 3 days, 10ng/ml liver cells are added into above-mentioned induction liquid for the long factor, 20ng/ml ~ 30ng/ml epidermal growth factor
Growth factor, 60ng/ml insulin-like growth factor-2s, after cultivating 10 days, mesenchymal stem cells differentiation is epithelial cell.
In order to further verify the effect that the present invention can generate, the cell plant piece prepared using the present invention has carried out ocular
Skin injury repair, detailed process include the following steps：
Step A, animal corneal limbal stem cell lacks eye surface diseases model and prepares：After new zealand white rabbit general anaesthesia, list is taken
Limbal area tissue is carefully rejected in branch hole local anaesthesia under surgical operation microscope（Diameter about 2mm）, reuse diameter 1.5cm
Circular filter paper piece pick 1M NaOH solutions, remove and be affixed on ocular pupil region after surplus liquid and remove for 15 ~ 30 seconds, using big
Measure normal saline flushing ocular；Postoperative daily use tobramycin eye drip 3 times, prevents from infecting；After 4 weeks, Ocular surface damage model
It prepares and completes, as shown in Figure 3A, in Fig. 3 A, it is seen that cornea is grown by a large amount of blood vessels, the opacity of the cornea；
Step B, Ocular surface healing：By above-mentioned animal pattern general anaesthesia, modeling branch hole is taken to implement local anaesthesia, in surgical operation microscope
Lower careful rejecting is covered in the conjunctival tissue, blood vessel and other scar tissues of ocular, fully exposes pupil region；By what is prepared
Cell plant piece basal layer is downward, is covered on cornea, and cell is planted piece and suture of conjunctiva using 8-0 sutures, is allowed to fixed
In ocular；Postoperative daily use tobramycin eye drip 3 times, continuous observation；Ocular surface healing situation is observed after 4 weeks, it is seen that ocular is damaged
Wound is repaired, and as shown in Figure 3B, most of blood vessel disappears, cornea start it is limpid, it is transparent.
The above is only presently preferred embodiments of the present invention, is not imposed any restrictions to the present invention, every according to the present invention
Technical spirit changes any simple modification, change and equivalent structure made by above example, still falls within skill of the present invention
In the protection domain of art scheme.
Claims (7)
1. a kind of cell for Ocular surface damage reparation plants piece, it is characterised in that：Including cell membrane lamella（1）And fibrin layer
（2）Double-layer structure, the cell membrane lamella（1）Thickness to be arranged to make up by 5~7 layers of epithelial cell is 50 μm~150 μm
Membrane structure, the fibrin layer（2）The loose porous film that thickness to be formed by Fibrin Glue is 200 μm~500 μm
Structure.
2. a kind of cell for Ocular surface damage reparation described in accordance with the claim 1 plants piece and preparation method thereof, feature exists
In：The epithelial cell is corneal epithelial cell, Buccal mucosa cell, amniotic epithelial cells and is divided by mescenchymal stem cell
One or more mixtures in the epithelial cell of change.
3. a kind of method for preparing cell as described in claim 1 and planting piece, which is characterized in that this approach includes the following steps：
Step 1: cell membrane lamella（1）Preparation, detailed process is：
Epithelial cell is seeded in progress cell patch culture in the cells Transwell by step 101；It is cultivated using DMEM high sugar
Base simultaneously changes once for every two days, and after cultivating 15~20 days, cell patch is formed；
Step 102 reduces the liquid level of DMEM high glucose mediums to cell membrane lamella（1）Highly, air-liquid interface culture is carried out, often
It changes a DMEM high glucose medium, and after 7 days, inverted microscope observation forms continuous epithelium layer, cell membrane lamella
（1）Culture terminates；
Step 2: fibrin layer（2）Preparation, detailed process is：
After DMEM high glucose mediums in step 201, removal Transwell cells, then remaining DMEM washed away using cleaning solution
High glucose medium；
Step 202 uses the CaCl that substance withdrawl syndrome is 40mmol/L2Solution compound concentration is the solidifying of 20U/ml~100U/ml
Hemase solution；
Step 203 prepares the fibrin original solution that quality-volumetric concentration is 0.02g/ml~0.1g/ml using pure water；Step
Rapid 204, the thrombin solution 0.5ml in step 202 is taken, the fibrin original solution 0.5ml in step 203, by fibrin ferment
Solution and fibrin original solution are uniformly applied to cell membrane lamella simultaneously（1）On, after waiting for 2min~5min, Fibrin Glue
Solidification, fibrin layer（2）Preparation terminates；
Step 3: the fibrin layer that will be prepared（2）With cell membrane lamella（1）Simultaneously from the bottom surface of the cells Transwell
It slowly takes off, has been made and has planted piece for the cell of Ocular surface damage reparation.
4. according to the method for claim 3, it is characterised in that：Transwell's cells described in step 101 is a diameter of
12mm, it is 2 × 10 that epithelial cell, which is seeded in the inoculum density in the cells Transwell,4A/cm2~1 × 105A/cm2。
5. according to the method for claim 3, it is characterised in that：In step 101 carry out cell patch culture when be in temperature
The CO for being 5% for 37 DEG C, mass percent concentration2It is carried out under atmospheric condition.
6. according to the method for claim 3, it is characterised in that：Contain matter in DMEM high glucose mediums described in step 101
Measure the fetal calf serum that percent concentration is 10%, the vitamin C that quality-volumetric concentration is 30mg/ml~70mg/ml, quality-body
The epidermal growth factor of a concentration of 2ng/ml~5ng/ml of product, the bovine brain that quality-volumetric concentration is 10 μ of μ g/ml~30 g/ml are hung down
Body extract and the sodium selenite that quality-volumetric concentration is 2ng/ml~5ng/ml.
7. according to the method for claim 3, it is characterised in that：Cleaning solution described in step 201 is phosphate buffer.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111657269A (en) * | 2020-06-22 | 2020-09-15 | 镇江雷音再生医学科技有限公司 | Method for protecting and treating fibrin glue before preservation of SMILE-derived human corneal lens |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1569259A (en) * | 2003-07-25 | 2005-01-26 | 北京以岭生物工程有限公司 | Tissue engineering autologous cornea epithelium and its preparation method |
| CN107137763A (en) * | 2017-05-04 | 2017-09-08 | 宁夏医科大学 | A kind of study of vascularized tissue engineering bone and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1569259A (en) * | 2003-07-25 | 2005-01-26 | 北京以岭生物工程有限公司 | Tissue engineering autologous cornea epithelium and its preparation method |
| CN107137763A (en) * | 2017-05-04 | 2017-09-08 | 宁夏医科大学 | A kind of study of vascularized tissue engineering bone and preparation method thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111657269A (en) * | 2020-06-22 | 2020-09-15 | 镇江雷音再生医学科技有限公司 | Method for protecting and treating fibrin glue before preservation of SMILE-derived human corneal lens |
| CN111657269B (en) * | 2020-06-22 | 2022-02-08 | 镇江雷音再生医学科技有限公司 | Method for protecting and treating fibrin glue before preservation of SMILE-derived human corneal lens |
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