CN106540325A - The method and cell transplantation composite and its application of cell culture and transplanting - Google Patents
The method and cell transplantation composite and its application of cell culture and transplanting Download PDFInfo
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- CN106540325A CN106540325A CN201510601759.XA CN201510601759A CN106540325A CN 106540325 A CN106540325 A CN 106540325A CN 201510601759 A CN201510601759 A CN 201510601759A CN 106540325 A CN106540325 A CN 106540325A
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Abstract
The invention discloses a kind of cell culture and implantation method, comprise the steps：1. the trace-etching-film for including one or more apertures is provided；2. one or more target cells are seeded on trace-etching-film, form cell transplantation composite；3. by the cell transplantation composite implantation of step 2 to skin or corneal wound.Additionally, the invention also discloses a kind of cell transplantation composite that builds of utilization tissue engineering technique, including the trace-etching-film in one or more apertures and one or more target cells, target cell is seeded on trace-etching-film.The cell transplantation composite is used for (being total to) of epithelial cell, melanocyte or other cells and cultivates, and realizes In vitro culture and the transplanting of cell, is required with the treatment met clinically to dermatosis and keratopathy.The present invention is workable, is that large area skin disappearance, heritability dermatosis, the regulation of depigmentation disease and skin color, and the treatment of keratopathy are provided a great convenience.
Description
Technical field
The invention belongs to biomedical sector, builds cell-trace-etching-film cell transplantation composite using tissue engineering technique, use
In epithelial cell (keratinocyte), melanocyte or other cells, (being total to) culture, realize the In vitro culture of cell
And transplanting.Present invention relates particularly to a kind of method of cell culture and transplanting.It is additionally related to one footpath of biocompatible cell
Mark etching-film cell transplantation composite and its application, to meet requirement clinically to treating skin disease.
Background technology
Cell transplantation is clinically widely used.The cell of transplanting can be many organization types, for example, skin, angle
The tissue of film, cartilage, bone, fat or other species.In practice, Skin Cell transplanting has very big clinical meaning, because
Expose outside for skin, be readily available.Various dermatosiss generally require skin transplantation.Particularly severe burn, it is difficult to which heal skin
Skin ulcer, vitiligo and some genetic dermatosis.Burn and skin ulcer are clinically very universal.Epithelial cell covers
Lid can promote the healing of wound.Vitiligo is a kind of acquired skin pigment depigmentation disease, shows as local or general property color
Plain depigmentation, is characterized with forming white macula, and histology and immunocytochemistry show that its skin lesion epidermal melanophore disappears.It can be with
Occur at any position of body.Vitiligo illness accounts for 0.5% to 4% in total population, is apt to occur in youngster.Genetic skin
Disease, such as epidermolysis bullosa, currently without particularly preferred therapy.China has 400～5,000,000 cornea sufferers every year
Person is waiting for corneal transplantation, but it is very few to contribute cornea person, causes patient's needs to wait as long for and can obtain suitable cornea,
Delay treatment.Corneal stroma pathological changes and the normal corneal blindness patient of corneal endothelium, such as keratoconuses, do not involve corneal endothelium
Corneal leukoma etc..These diseases can be treated with deep lamellar keratoplasty (deep lamellar keratoplasty, DLKP),
But corneal donor limited source.
One of cell therapy focuses on autogenous cell transplantation.The basic principle is to derive what is needed from the health tissues of patient
Cell, cultivates them in vitro, then is transplanted to the acceptor regions of correspondingly same patient.Skin epithelial cell is implanted in clinic
Can be used for treating burn and skin ulcer, but clinical effectiveness is not proved to be entirely satisfactory.It is leukodermic to be characterised by skin
Interior melanocyte loss.Traditional treatment meanss include that endo-medicine, ultraviolet are irradiated with surgical operation therapy etc..Medicine
The cure rate for the treatment of and ultraviolet irradiation is limited.Surgical operation therapy method mainly includes AUTOEPIDERMIC GRAFTING, melanocyte suspension
Transplanting etc..It is effective that these Therapeutic Method confirm.But, surgical operation therapy is clinically carried out does not have universality.Heredity
Property the current also no particularly preferred therapy of dermatosis.
Current method yet unresolved issue includes：Epidermic grafting needs large area bark fetching, and cell suspension in cell suspension transplanting
It is easy to run off, affects the treatment, and is not suitable for large area transplanting；Undifferentiated epidermis cell grafting lazy weight；How from tissue training
The epidermal graft of full wafer is obtained in foster container；The epidermal graft of culture is shunk in migration process, is partially destroyed；Patient is available for skin
Position lacks；For the position cicatrization of skin.The method of the present invention allows these and other relevant issues to minimize or be resolved.
Trace-etching-film (nucleopore membranes) is a kind of new material.It is banged using the energetic ion produced by heavy ion avcceleration
Thin dielectric film is hit, and partial modification is produced near the track passed through, micropore is produced by chemical etching then.Pore size
Can be controlled by the size for controlling high-energy heavy ion beam.Trace-etching-film is any biological inert in itself, there is no film itself right
The pollution of cell.The purposes of existing disclosed trace-etching-film is usually as the instrument filtered with sieve particle.There is no for people
Body skin epithelial cell (keratinocyte), human body epithelial stem cell, corneal epithelial cell, corneal epithelial stem cells, into fibre
Dimension cell, human body melanocyte, the open report of the culture and transplanting of human body melanoblast and human body melanocyte stem cells.
The content of the invention
One of the technical problem to be solved in the present invention is for the deficiencies in the prior art, there is provided the side of a kind of cell culture and transplanting
Method, cell culture technology and biomaterial trace-etching-film grafting device are combined by which, improve cell from culture with trace-etching-film
Ware is damaged to during skin or corneal transplantation, and the growth of epithelial cell and melanocyte is adjusted using somatomedin, is transplanted
Epithelial cell, epithelial stem cell, melanocyte and melanocyte stem cells are treating various dermatosis and keratopathy.
The two of the technical problem to be solved in the present invention are to provide a kind of cell transplantation composite.
The three of the technical problem to be solved in the present invention are the application for providing the cell transplantation composite.
The technical problem to be solved in the present invention is achieved through the following technical solutions:
In one aspect of the invention, there is provided a kind of cell culture and implantation method, comprise the steps：
(1) trace-etching-film for including one or more apertures is provided；
(2) one or more target cells are seeded on trace-etching-film, form cell transplantation composite；
(3) by the cell transplantation composite implantation of step (2) to skin or corneal wound.
As currently preferred technical scheme, in step (1), the size in the aperture of the trace-etching-film is that 0.01-5 is micro-
Rice；The trace-etching-film adopts one or more of following polymer：Poly- Polycarbonates (Merlon),
Polyethylene terephthalate (polyethylene terephthalate), Polyimide (polyimides), CR-39 (are learned
Name carbon this acid propylene acetic acid, or claim allyl diglycol acid fat (Dially Glycol Carbonates), it is most widely used life
Produce the material of ordinary resin eyeglass), or allyl diglycol carbonate (allyl diglycol carbonates),
Polyvinylidene fluoride (polyvinylidene fluoride), Poly (methyl methacrylate) (poly- (methacrylic acid
Methyl ester) (PMMA), Polypropylene (polypropylene).In one embodiment, cell transplantation device is Merlon and pi-allyl
Diglycol carbonates.In one embodiment, trace-etching-film is from Whatman (Kent, UK), EMD Millipore
(Billerica, Massachusetts), Membrane solutions (Plano, Texas), or it4ip (Seneffe,
Belgium).Polycarbonate membrane and allyl diglycol carbonates film can be implanted in Mus, without harmful effect.
As currently preferred technical scheme, in step (1), the sterilized sterilizing of the trace-etching-film, the sterilization
Concretely comprise the following steps：The trace-etching-film volumetric concentration is 75% alcohol-pickled 6-24 hours, is cleaned followed in turn by PBS residual
Ethanol is stayed, is placed under uviol lamp, irradiating 3-24 hours.
As currently preferred technical scheme, in step (1), after sterilization is completed, according to skin or corneal damage portion
The size of position, trace-etching-film be cut into than big 0.1-1 centimetre of damaged part shape (biomaterial trace-etching-film grafting device
Size can be determined according to receptor wound or damaged part size), cell culture fluid is injected in Tissue Culture Plate, is placed in thin
In the CO that 37 DEG C, volumetric concentration are 5% in born of the same parents' incubator2, saturated humidity condition cultivated.
As currently preferred technical scheme, increase following steps before step (1)：Surface to the trace-etching-film
Processed, including tissue culture treated, poly- D-Lys coating surface, plus the compositionss that can be transformed into gel phase from liquid are applied,
To increase the attachment and growth of cell；Said composition does not have lethal or toxic action to somatic cell；The compositionss can include one
Individual or multiple extracellular matrix (ECM) compositions (for example, the basement membrane of restructuring), and it is other process with increase cell attachment and
Growth.
As currently preferred technical scheme, increase following steps between step (1) and step (2)：In the track
The surface of etching-film, plus apply one or more compounds that can be administered to receptor；One or more of compounds are selected from as follows：
Somatomedin, extracellular matrix protein, enzyme, reporter molecule, liposome and nucleic acid.The somatomedin can be epidermis life
The long factor (EGF), platelet derived growth factor (PDGF), keratinocyte growth factor (KGF), Desmocyte growth factor
Sub (FGF) or transforming growth factor (TGF), thrombin, ET-3 (ET-3), the extracellular matrix protein can be
Collagen protein, laminin，LN, fibrin or sulphuric acid second look down heparin etc..
As currently preferred technical scheme, in step (2), the one kind of one or more of target cells selected from following cell
Or multiple combination：Epithelial cell, melanocyte and fibroblast；The epithelial cell includes skin and hair follicle (hair)
Epithelial cell, skin and hair follicle (hair) epithelial stem cell, corneal epithelial cell, corneal epithelial stem cells；The melanin
Cell includes the melanocyte stem cells that the melanocyte of skin-derived, hair follicle (hair) originate, melanoblast, black
Plain cell；The epithelial cell, melanocyte can from other cell transformations including pluripotent stem cell or myeloid-lymphoid stem cell,
Such as pluripotent stem cell or myeloid-lymphoid stem cell；The culture of cell can be the 3D cultures of monolayer, or multilamellar.
As currently preferred technical scheme, in step (2), the target cell can be fetal cell or other it is autologous or
Person allosome stem cell (for example, corneal stem cells, endothelial stem cell or mescenchymal stem cell).
Used as currently preferred technical scheme, step (2) is specially：With 103-l08Individual target cell is seeded in trace-etching-film
On, according to sick human needs, cell attachment can be waited directly to use after trace-etching-film or add cell culture fluid, be placed in
Cell culture incubator, incubation time are 8 hours -30 days, and a cell culture fluid was changed per two to three days, cell amplification with
After reuse acquisition cell transplantation composite.
It is before methods described is additionally may included in the implantation step, prolonged to cultivate, one or more target cells and footpath
After directly adhering in mark etching-film grafting device, cell significantly need not be bred.After cell adhesion, cell-trace-etching-film is moved
Plant device to be used for being transplanted.
As currently preferred technical scheme, in step (3), the skin or corneal wound after treatment, cell transplantation
To skin or corneal wound, cell one is arrived composite implantation facing to wound, the cell migration on cell transplantation composite
On wound.
In another aspect of this invention, there is provided a kind of cell transplantation composite, including the trace-etching-film in one or more apertures
With one or more target cells, the target cell is seeded on trace-etching-film.
Used as currently preferred technical scheme, the size in the aperture of the trace-etching-film is 0.01-5 microns；The track
Etching-film adopts one or more of following polymer：Merlon, polyethylene terephthalate, polyimides, CR-39,
Allyl diglycol carbonates, polyvinylidene fluoride, polymethyl methacrylate, polypropylene；One or more of target cells
One or more selected from following cell is combined：Epithelial cell, melanocyte and fibroblast；The target cell is fetus
Cell or allosome adult stem or allosome adult cell.
In another aspect of this invention, there is provided above-mentioned cell transplantation composite is preparing the product for the treatment of dermatosis and keratopathy
Application in product.
Compared with the prior art, the beneficial effects of the present invention is：Epithelial cell, (skin keratin forms cell or angle to the present invention
Film epithelial cell), or melanocyte, or epithelial cell and melanocyte, or epithelial cell, melanocyte and into fibre
Dimension cell co-cultivation and track etching film combination, culture and implantation method on trace-etching-film, such method can be used to
The time of cell culture is reduced, allows cell not to be damaged in migration process, increase the efficiency of cell transplantation.And unnecessary is thin
Born of the same parents can be retained, and cold preservation is used for doing later treatment.
The present invention combines cell culture technology and biomaterial, and cell is bred on trace-etching-film (nucleopore membranes) biomaterial,
Formed cell composite-cell transplantation device, another position of cell composite implantation to body carry out the method for cell therapy and
Material.This is a kind of method of novel manufacture cell and track etching film composite material.The present invention is described for increasing from table
Cell, cell propagation are extracted in skin, scalp, hair and cornea, and is transplanted to the method and material at another position.This method
Including cell from epidermis, scalp, is separated in hair and cornea, the target cell that wherein at least some is separated can be lost in track
Breed on engraved film, and and then target cell in grafting device is transplanted to another skin part or cornea to reach cell transplantation and thin
The effect of born of the same parents' treatment.
The present invention selects the good trace-etching-film of biocompatibility, by the size for regulating and controlling track etching membrane aperture, it is easy to cell
Culture and fungi-proofing property holding, to reach the purpose of bio-safety.Meanwhile, the present invention can on Tissue Culture Plate direct construction
Carrier material, it is to avoid loaded down with trivial details post processing and the secondary pollution problem such as cut out to carrier material again.The carrier material that Jing cultures are processed,
With superior mechanical performance, fungi-proofing performance meets and wanting of shifting for cell and transplant directly can be taken out from Tissue Culture Plate
Ask.Additionally, the present invention is realized by the inoculum density of regulation cell, incubation time and cultural method, particularly somatomedin
Regulation and control to Human Epithelial Cells, epithelial stem cell, melanocyte, melanoblast and melanocyte stem cells, to difference
Individual demand, makes epithelial cell and the melanocytic both effectiveness expression succeeded in transplanting, with the operable of reality
Property, be large area skin disappearance, heritability dermatosis, the regulation of depigmentation disease and skin color and corneal damage treatment carry
Great convenience is supplied.
Unless otherwise defined, all technologies used herein and scientific terminology with by ordinary skill of the art
The identical implication that personnel are generally understood.Although or the method that be equal to similar with those described herein and material can be used for
Implement the present invention, but suitable method and material will be illustrated below.All publications, patent application mentioned by herein,
Entire contents are all expressly incorporated herein by patent and other lists of references by way of reference.In the case of a conflict, with this explanation
Book (including definition) is defined.Additionally, the material, method and example are merely illustrative, it is not intended that limited.
Description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description：
Fig. 1 is the high power photo schematic diagram of trace-etching-film in the embodiment of the present invention 7；
Fig. 2 is that the cell in the embodiment of the present invention 7 is bred trend in the track etching membrane aperture of 0.01-3 microns substantially and kept
The scanned photograph schematic diagram of good form；
Fig. 3 be when cell is inoculated in trace-etching-film in the embodiment of the present invention 7 around culture dish cell situation schematic diagram；
Fig. 4 is cell undergrowth on the medical silicon carrier timbering material of 20% formic acid deposition pretreatment in the embodiment of the present invention 7
Schematic diagram；
Fig. 5 is schematic diagram of the cell migration to culture dish surface on trace-etching-film cell transplantation device in the embodiment of the present invention 7.
Fig. 6 is that after trace-etching-film cell transplantation device is placed on skin wound in the embodiment of the present invention 1, skin completes the schematic diagram that heals.
In Fig. 6, it is around original skin, central is the skin (arrow points to the skin of healing) of new healing.
Fig. 7 is that after trace-etching-film cell transplantation device is placed on skin wound in the embodiment of the present invention 4, melanocyte moves to skin
Inside skin and hair follicle, skin heal completely after schematic diagram.Wherein, in Fig. 7 A, skin at trace-etching-film cell transplantation device implantation
It is black (arrow indication), surrounding skin is white, has black hair to grow (the head indication of arrow).In Fig. 7 B,
Histology's (H＆E dyeing), arrow point to the hair for having black.In Fig. 7 C, the dyeing of Fontana-Mason melanin, arrow
Sensing has the hair of black, and hair surface also has melanin.
Fig. 8 is that after trace-etching-film cell transplantation device is placed on skin wound in the embodiment of the present invention 5, melanocyte moves to skin
Inside skin and hair follicle, skin organizes the schematic diagram after the dyeing of Fontana-Mason melanin after healing completely.Wherein, in Fig. 8 A
Hair around at trace-etching-film cell transplantation device implantation, no melanin.Trace-etching-film cell transplantation device implantation in Fig. 8 B
The following hair in place, has melanin (arrow is pointed to melanin).In Fig. 8 C at the implantation of trace-etching-film cell transplantation device around
Subcutaneous tissue, no melanin.In Fig. 8 D, subcutaneous tissue following at the implantation of trace-etching-film cell transplantation device, has melanin
(arrow is pointed to melanin).
Specific embodiment
Below example facilitates a better understanding of the present invention, but does not limit the present invention.Experimental technique in following embodiments,
If no special instructions, it is conventional method.In following embodiments, test material used, if no special instructions, is conventional life
Change what Reagent Company was commercially available.
Embodiment 1
At 0.02 micron, trace-etching-film adopts Merlon and allyl diglycol for the size control in the aperture of trace-etching-film
Carbonic ester；Before trace-etching-film is used for inoculating cell, with alcohol-pickled 12 hours that concentration is 75%, wash followed in turn by PBS
Net residual ethanol, is placed in irradiating 8 hours under uviol lamp.After completing sterilization, according to the size of skin lesion, track is lost
Engraved film is cut into 0.1 centimetre bigger than skin lesion of shape, and cell culture fluid is injected in Tissue Culture Plate, be placed in cell culture incubator in
37 DEG C, the CO that volumetric concentration is 5%2, saturated humidity condition cultivated.Epithelial cell (the cutin shape come by skin and hair follicle
Into cell) with l08Individual cell is seeded on trace-etching-film, while adding 100 milliliters of cell culture fluid, is placed in cell culture
Case.A cell culture fluid was changed per two days, incubation time is 10 days, that is, obtain epithelial cell-track etching film composite material
(trace-etching-film grafting device).Growth of the epithelial cell on film can be monolayer, or multi-layer cellular.Work as use
During the culture fluid of the three-dimensional differentiation of epithelial cell, during the CnT-Prime 3D Barrier Medium of such as CELLNTEC, epithelium
Cell can grow up to the cell of multilamellar as skin.Clinically can be used to treat large area scald, skin ulcer.
C57BL/6 black mouse skin horny layer pancreatin is digested to individual cells suspension, cell culture in trace-etching-film
On, contain S-MEM, the serum of 8% (vol/vol) chelexed, 1% Pen .- Strep, after 10 days in culture fluid
Form epithelial cell-track etching film composite material.The source of epithelial cell can also be from pluripotent stem cell (multipotent
Stem cells) and myeloid-lymphoid stem cell (pluripotent stem cells) differentiate.In nude mice zoopery, in nude mice
Back cuts the skin lesion of 1 cm diameter.Nude mice skin wound through hemostasis process after, cell-track etching film composite material (footpath
Mark etching-film grafting device) wound is transplanted to, cell one is wrapped up film on wound, is transplanted in trace-etching-film facing to wound
Cell propagation and migration on device.After 10-14 days, wound heals (see Fig. 6, arrow points to the skin of healing) completely.
Embodiment 2
The size in the aperture of trace-etching-film is controlled at 2 microns, and trace-etching-film is from Whatman (Kent, UK), EMD
Millipore (Billerica, Massachusetts), Membrane solutions (Plano, Texas), or it4ip
(Seneffe,Belgium).Before trace-etching-film is used for inoculating cell, with alcohol-pickled 12 hours that volumetric concentration is 75%,
Residual ethanol is cleaned followed in turn by PBS, is placed in irradiating 3 hours under uviol lamp.After completing sterilization, according to the big of skin lesion
It is little, trace-etching-film is cut into 1 centimetre bigger than skin lesion of type shape, cell culture fluid is injected in Tissue Culture Plate, cell is placed in
In the CO that 37 DEG C, volumetric concentration are 5% in incubator2, saturated humidity condition cultivated.The epithelium that skin or hair follicle come is done
Cell (cutin forms stem cell) expression CD200, can separate epithelial stem cell according to this, the epithelium come by skin or hair follicle
Stem cell (cutin formed stem cell) is with 104Individual cell is seeded on trace-etching-film, while adding 100 milliliters of cell culture
Liquid, is placed in cell culture incubator.Per two days change a cell culture fluid, incubation time be 8 hours, that is, obtain epithelial stem cell-
Track etching film composite material (trace-etching-film grafting device).Clinically can be used to treat intractable skin ulcer, heritability
Dermatosis, the such as treatment to epidermolysis bullosa.
We are digested to individual cells suspension, cell surface protein CD34 the cell pancreatin that C57BL/6 black mouse hair follicles come
Can be used for the epithelial stem cell of the Mus of identification with α 6-integrin, epithelial stem cell is screened with cell flow cytometer,
Cell culture contains DMEM on trace-etching-film, in culture fluid:F12(3:1) culture fluid, 2mM glutamine, 15% (vol/vol)
The serum of chelexed, 1% Pen .- Strep, 100ng/ml cholera toxins, 100ng/ml hydrodotisone, 8 is little
When after formed cell-track etching film composite material.The source of epithelial stem cell can also be from pluripotent stem cell (multipotent
Stem cells) and myeloid-lymphoid stem cell (pluripotent stem cells) differentiate.In nude mice zoopery, in nude mice
Back cuts the skin lesion of 1 cm diameter.Nude mice skin wound through hemostasis process after, cell-track etching film composite material (footpath
Mark etching-film grafting device) wound is transplanted to, cell one is wrapped up film on wound, is transplanted in trace-etching-film facing to wound
Cell on device is bred and is moved on wound.After 10-14 days, wound heals completely.
Embodiment 3
At 1 micron, trace-etching-film adopts Merlon and allyl diglycol carbonic acid for the size control in the aperture of trace-etching-film
Ester；Before trace-etching-film is used for inoculating cell, with alcohol-pickled 12 hours that volumetric concentration is 75%, wash followed in turn by PBS
Net residual ethanol, is placed in irradiating 24 hours under uviol lamp.After completing sterilization, according to the size of corneal injury, footpath
Mark etching-film is cut into the type shape than damaging big 0.2 centimetre, injects cell culture fluid, be placed in cell culture incubator in Tissue Culture Plate
In in the CO that 37 DEG C, volumetric concentration are 5%2, saturated humidity condition cultivated.Corneal epithelial cell or corneal epithelium are done
Cell is with 104-l06Individual cell is seeded on trace-etching-film, while adding 100 milliliters of cell culture fluid, is placed in cell training
Foster case.A cell culture fluid was changed per two days, incubation time is 10 days, that is, obtain corneal epithelial cell-trace-etching-film multiple
Condensation material obtains corneal epithelial stem cells-track etching film composite material (trace-etching-film grafting device).Corneal epithelial cell
Or the source of corneal epithelial stem cells can also be from pluripotent stem cell (multipotent stem cells) and myeloid-lymphoid stem cell
(pluripotent stem cells) is differentiated.
Clinically can be used to treat corneal stroma pathological changes and the normal corneal blindness patient of corneal endothelium, such as keratoconuses, do not tire out
And the corneal leukoma of corneal endothelium, it is muddy caused by corneal injury and pathological changes.Cell growth factor can be it is single, or appoint
What combination.The growth of epithelial cell can be with monolayer, or multilamellar.When the culture using the three-dimensional differentiation of epithelial cell
During liquid, during the CnT-Prime 3D Barrier Medium of such as CELLNTEC, epithelial cell can grow up to as skin
The cell of multilamellar.Impaired cornea after treatment, (transplant corneal epithelial cell-track etching film composite material by trace-etching-film
Device) wound is transplanted to, cell one is on corneal wound, the cell migration in trace-etching-film grafting device to corneal wound.
Film is fixed and is wrapped up on wound, corneal healing after 14-21 days.
Embodiment 4
The size in the aperture of trace-etching-film is controlled at 1 micron, and trace-etching-film is from Whatman (Kent, UK), EMD
Millipore(Billerica,Massachusetts)、Membrane solutions(Plano,Texas)、
Or it4ip (Seneffe, Belgium).Before trace-etching-film is used for inoculating cell, with the ethanol leaching that volumetric concentration is 75%
Bubble 6 hours, cleans residual ethanol followed in turn by PBS, is placed in irradiating 10 hours under uviol lamp.After completing sterilization, according to
The size of skin lesion, is cut into 0.3 centimetre bigger than skin lesion of type shape trace-etching-film, cell culture fluid is injected in Tissue Culture Plate,
It is placed in cell culture incubator in the CO that 37 DEG C, volumetric concentration are 5%2, saturated humidity condition cultivated.Skin or hair follicle are come
Melanocyte or melanoblast, melanocyte stem cells are with 103-l05Individual cell is seeded on trace-etching-film, while plus
Enter the cell culture fluid of 100-200 milliliters, be placed in cell culture incubator.A cell culture fluid was changed per three days, incubation time is
30 days, i.e. acquisition melanocyte-track etching film composite material, melanoblast-track etching film composite material, or it is black
Pigment stem cell-track etching film composite material (trace-etching-film grafting device).After skin vitiligo position epidermis grinds off, black
Plain cell-track etching film composite material or melanoblast-track etching film composite material, or melanocyte stem cells-track erosion
, to wound, cell one is on wound, the cell migration in trace-etching-film grafting device to wound for engraved film composite implantation.
The cell pancreatin that C57BL/6 black mouse hair follicles come is digested to individual cells suspension, cell culture in trace-etching-film
On, cell culture fluid contains MEM, and the NaHCO 3 of 5%FBS, 5mM is washed, 25 μM of β mercaptoethanols, the bar of 200nM
Bean alcohol -12- myristate -13- acetass, 1 × non essential amino acid, 1 × Sodium Pyruvate, 1 × penicillin/streptomycin, melanin
Cell is bred in culture fluid, and melanocyte-track etching film composite material is formed after 30 days.Melanocyte, melanin are female
The source of cell or melanocyte stem cells can also be from pluripotent stem cell (multipotent stem cells) and myeloid-lymphoid stem cell
(pluripotent stem cells) is differentiated.In nude mice zoopery, the skin lesion of 1 cm diameter is cut in nude mice back.
Nude mice skin wound after hemostasis process moves melanocyte-track etching film composite material (trace-etching-film grafting device)
Wound is planted, cell one is wrapped up film on wound facing to wound, and the cell in trace-etching-film grafting device is bred and moved
Move on on wound.After 10-14 days, wound heals completely, and have in the hair follicle in wound many melanocytes (see Fig. 7 A,
Fig. 7 B and Fig. 7 C).Clinically can be used to treat vitiligo and other pigments lack disease.
Embodiment 5
At 0.01 micron, trace-etching-film adopts Merlon and allyl diglycol carbon for the size control in the aperture of trace-etching-film
Acid esters；Before trace-etching-film is used for inoculating cell, with alcohol-pickled 24 hours that volumetric concentration is 75%, clean followed in turn by PBS
Residual ethanol, is placed in irradiating 8 hours under uviol lamp.After completing sterilization, according to the size of skin lesion, trace-etching-film
Be cut into 0.5 centimetre bigger than skin lesion of type shape, cell culture fluid injected in Tissue Culture Plate, be placed in cell culture incubator in 37 DEG C,
Volumetric concentration is 5% CO2, saturated humidity condition cultivated.The epithelial cell (keratinocyte) come by skin or hair follicle
Mix with melanocyte with 105-l06Individual cell is seeded on trace-etching-film, while adding the cell culture of 10-200 milliliters
Liquid, is placed in cell culture incubator.A cell culture fluid was changed per two days, incubation time is 15 days, that is, obtain epithelium and melanin
Cell-track etching film composite material (trace-etching-film grafting device).Clinically can be used to treat large area scald, skin
Ulcer, skin injury, genetic dermatosis and vitiligo.The growth of epithelial cell can be with monolayer, or multilamellar.
When the culture fluid using the three-dimensional differentiation of epithelial cell, during the CnT-Prime 3D Barrier Medium of such as CELLNTEC,
Epithelial cell can grow up to the cell of multilamellar as skin.The epithelial cell come by C57BL/6 black mouse skin and hair follicle and black
Plain cell pancreatin is digested to individual cells suspension, cell culture on trace-etching-film, containing S-MEM in culture fluid, 8%
(vol/vol) serum of chelexed, 1% Pen .- Strep, 100nM melanophore hormones, after 15 days formed cell-
Track etching film composite material.The source of epithelium and melanocyte can also be from pluripotent stem cell (multipotent stem
Cells) differentiate with myeloid-lymphoid stem cell (pluripotent stem cells), the mixed culture of the above can also be melanin
Cell, fibroblast and keratinocyte co-cultivation.
In nude mice zoopery, the skin lesion of 1 cm diameter is cut in nude mice back.Nude mice skin wound through hemostasis process after,
Cell-track etching film composite material (trace-etching-film grafting device) is transplanted to wound, and cell one is wrapped up film facing to wound
On wound, the cell in trace-etching-film grafting device is bred and is moved on wound.After 10-14 days, wound heals completely,
And in the subcutaneous and hair follicle in wound, have melanocyte, but be remote from the skin of trace-etching-film grafting device and there is no melanin
(see Fig. 8 A-D, arrow points to the hair and subcutaneous melanin of black, Fontana-Mason dyeing).
Embodiment 6
The size in the aperture of trace-etching-film is controlled at 5 microns, and trace-etching-film is from Whatman (Kent, UK), EMD
Millipore (Billerica, Massachusetts), Membrane solutions (Plano, Texas), or
it4ip(Seneffe,Belgium).It is before trace-etching-film is used for inoculating cell, little with alcohol-pickled 12 that volumetric concentration is 75%
When, residual ethanol is cleaned followed in turn by PBS, be placed in irradiating 8 hours under uviol lamp.After completing sterilization, according to skin lesion
Size, trace-etching-film is cut into 0.8 centimetre bigger than skin lesion of shape, cell culture fluid is injected in Tissue Culture Plate, is put
In the CO that 37 DEG C, volumetric concentration are 5% in cell culture incubator2, saturated humidity condition cultivated.Need skin or
The epithelial cell and melanocyte that hair follicle comes mixes with 104-l06Individual cell is seeded on trace-etching-film, while adding 100 millis
The cell culture fluid for rising, waits cell adhesion on film, it is not necessary to had significant proliferation, that is, obtain epithelial cell-trace-etching-film composite wood
Material (trace-etching-film grafting device).Clinically can be used to treat large area scald, skin ulcer and vitiligo.C57BL/6
The epithelial cell and melanocyte pancreatin that black mouse skin and hair follicle come is digested to individual cells suspension, and cell culture is existed
On trace-etching-film, containing S-MEM in culture fluid, the serum of 8% (vol/vol) chelexed, 1% Pen .- Strep,
100nM melanophore hormones, after 12 hours, cell attachment forms cell-track etching film composite material.The source of epithelial cell
Can also be from pluripotent stem cell (multipotent stem cells) and myeloid-lymphoid stem cell (pluripotent stem cells)
Differentiate.
In nude mice zoopery, the skin lesion of 1 cm diameter is cut in nude mice back.Nude mice skin wound through hemostasis process after,
Cell-track etching film composite material (trace-etching-film grafting device) is transplanted to wound, cell one facing to wound, film bag
Prick on wound, the cell in trace-etching-film grafting device is bred and moved on wound.After 10-14 days, wound is healed completely
Close, and have melanocyte in the hair follicle in wound.
Embodiment 7
Studied as the trace-etching-film timbering material to obtained by, with even porous structure (see Fig. 1), be adapted to cell life
It is long.Simultaneously there is good mechanical performance, preparing, load, transfer and migration process do not occur broken and rupture, meet and turn
The requirement for moving and transplanting.In the building process of cell-trace-etching-film cell transplantation device composite, cell is in biocompatibility footpath
Form on mark etching-film cell transplantation device is good (see Fig. 2), and the structure of porous benefits the renewal of culture fluid, thin so as to promote
The propagation of born of the same parents, makes cell keep good activity (see Fig. 3) similar with the cell grown on surrounding culture dish.And cell is at which
His timbering material, such as deposits pretreatment medical silicon carrier (Eves PC et al, J by 20% formic acid of plasma polymerization
Invest Dermatol.200,1554-64) on undergrowth, cell depends on and breeds bad (see Fig. 4).When track is lost
After the upset of engraved film cell transplantation device, what the cell on trace-etching-film cell transplantation can be good is transplanted to following culture from film
Ware surface (see Fig. 5), it was demonstrated that the function of trace-etching-film cell transplantation device composite sertoli cell transplanting.Build biofacies
Epithelial cell and melanocyte culture are formed cell skin graft on support and reach culture and transfer integration, can be had by capacitive support
Effect improves the weak point of traditional suspension transplantation therapy.
Claims (13)
1. a kind of cell culture and implantation method, it is characterised in that：Comprise the steps：
(1) trace-etching-film for including one or more apertures is provided；
(2) one or more target cells are seeded on trace-etching-film, form cell transplantation composite；
(3) by the cell transplantation composite implantation of step (2) to skin or corneal wound.
2. the method for claim 1, it is characterised in that in step (1), the aperture of the trace-etching-film it is big
It is little for 0.01-5 microns；The trace-etching-film adopts one or more of following polymer：Merlon, poly- terephthaldehyde
Sour glycol ester, polyimides, CR-39, allyl diglycol carbonates, polyvinylidene fluoride, polymethyl methacrylate,
Polypropylene.
3. the method for claim 1, it is characterised in that in step (1), the sterilized sterilizing of the trace-etching-film,
The sterilization is concretely comprised the following steps：The trace-etching-film volumetric concentration is 75% alcohol-pickled 6-24 hours, subsequently again
Residual ethanol is cleaned with PBS, is placed under uviol lamp, irradiating 3-24 hours.
4. the method for claim 1, it is characterised in that in step (1), after sterilization is completed, according to skin
Skin or the size at corneal damage position, are cut into the shape than big 0.1-1 centimetre of damaged part trace-etching-film, in cell culture
Cell culture fluid is injected in plate, is placed in cell culture incubator in the CO that 37 DEG C, volumetric concentration are 5%2, saturated humidity condition enters
Row culture.
5. the method for claim 1, it is characterised in that increased following steps before step (1)：To the track
The surface of etching-film is processed, including tissue culture treated, poly- D-Lys coating surface, plus apply can be transformed into from liquid it is solidifying
The compositionss of glue phase, to increase the attachment and growth of cell；Said composition does not have lethal or toxic action to somatic cell；Described group
Compound includes one or more extracellular matrix components.
6. the method for claim 1, it is characterised in that increase following steps between step (1) and step (2)：
On the surface of the trace-etching-film, plus apply one or more compounds that can be administered to receptor；One or more of chemical combination
Thing is selected from as follows：Somatomedin, extracellular matrix protein, enzyme, reporter molecule, liposome and nucleic acid.
7. the method for claim 1, it is characterised in that in step (2), one or more of target cells are selected from such as
One or more combination of lower cell：Epithelial cell, melanocyte and fibroblast；The epithelial cell include skin and
Hair follicle epithelial cells, skin and follicular epithelium stem cell, corneal epithelial cell, corneal epithelial stem cells；The melanocyte
The melanocyte stem cells in melanocyte, the hair follicle source including skin-derived, melanoblast, melanocyte；It is described
Epithelial cell, melanocyte can be from other cell transformations including pluripotent stem cell or myeloid-lymphoid stem cell；The culture of cell
Can be the 3D cultures of monolayer, or multilamellar.
8. the method for claim 1, it is characterised in that in step (2), the target cell are fetal cells or dry
Cell.
9. the method for claim 1, it is characterised in that step (2) is specially：With 103-l08Individual target cell inoculation
On trace-etching-film, according to sick human needs, cell attachment can be waited directly to use after trace-etching-film or add cell
Culture fluid, is placed in cell culture incubator, and incubation time is 8 hours -30 days, and a cell culture fluid was changed per two to three days,
Acquisition cell transplantation composite is reused after cell amplification.
10. the method for claim 1, it is characterised in that in step (3), the skin or wound after treatment,
To skin or corneal wound, cell one is facing to wound, thin on cell transplantation composite for cell transplantation composite implantation
Born of the same parents are moved on wound.
A kind of 11. cell transplantation composites, it is characterised in that trace-etching-film including one or more apertures and one or
Multiple target cells, the target cell are seeded on trace-etching-film.
12. cell transplantation composites as claimed in claim 11, it is characterised in that the aperture of the trace-etching-film it is big
It is little for 0.01-5 microns；The trace-etching-film adopts one or more of following polymer：Merlon, poly- terephthaldehyde
Sour glycol ester, polyimides, CR-39, allyl diglycol carbonates, polyvinylidene fluoride, polymethyl methacrylate,
Polypropylene；One or more combination of one or more of target cells selected from following cell：Epithelial cell, melanocyte and
Fibroblast；The target cell is fetal cell or stem cell.
The 13. cell transplantation composites as described in claim 11 or 12 are preparing the product for the treatment of dermatosis and keratopathy
Application in product.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113518655A (en) * | 2019-01-30 | 2021-10-19 | 瑞普利金公司 | Method for filtering biological fluids using track-etched membranes |
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| CN100998526A (en) * | 2006-01-10 | 2007-07-18 | 上海组织工程研究与开发中心 | Corneal graft |
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