CN101629162B - Tissue engineering cell piece and preparation method thereof - Google Patents
Tissue engineering cell piece and preparation method thereof Download PDFInfo
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- CN101629162B CN101629162B CN2009100421476A CN200910042147A CN101629162B CN 101629162 B CN101629162 B CN 101629162B CN 2009100421476 A CN2009100421476 A CN 2009100421476A CN 200910042147 A CN200910042147 A CN 200910042147A CN 101629162 B CN101629162 B CN 101629162B
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Abstract
The invention belongs to the field of tissue engineering, in particular to a cell sheet engineering and a preparation method thereof. The cell sheet is prepared with the following methods: firstly, an electron accelerator is used for radiating on the surface of the cell culture supporter for synthesizing poly-N-isopropylacrylamide; then, temperature is controlled to cause the poly-N-isopropylacrylamide to be hydrophobicity phase, and cells are cultured on the phase; temperature is changed to cause hydrogel to be hydrophilic phase; with the method that a polymeric membrane is used for adsorbing or a clamp is used for clamping to separate the cell sheet engineering. Obtained cell sheets are cultivated by multiple stratification, or protein adhering liquid is used for adhering to obtain multiple layers of cell sheets. The cell sheet obtained by the invention can keep desmosome structure between cells, ZO-1 tight connection structure and the connection structure between other cells, can keep basement membrane protein and a plurality of components thereof relative to adhesion, such as integrin and the like and has certain strength.
Description
Technical field
The invention belongs to field of tissue engineering technology, be specifically related to a kind of cell sheet engineering of obtaining and preparation method thereof of cultivating by temperature sensitive property material-N N-isopropylacrylamide hydrogel.
Background technology
Poly--N N-isopropylacrylamide (poly (N-isopropylacrylamide), PIPAAm) hydrogel is the temperature responsive intelligent polymkeric substance, it is in a certain temperature, just usually said lowest critical solution temperature (lower critical solutiontemperature, LCST) exist the discontinuous volume of reversible to change mutually near, the LCST of PNIPAAm hydrogel is about 32 ℃.This material has very application prospects in fields such as machinery, chemistry and biological medicines.Its application in field of tissue engineering technology in recent years more and more is subject to people's attention, because the existence of LCST, it has outstanding effect when being used as cell cultures.This be because, when envrionment temperature was higher than LCST slightly, PIPAAm hydrogel volume can suddenly violently shrink, and is hydrophobic at this moment, with cell seeding thereon, cell can tactophily; When envrionment temperature drops to LCST when following, hydrogel is swelling again, and this moment, hydrogel was hydrophilic, and cell is just from its surface isolation.So we can be when hydrogel be hydrophobic inoculating cell, cultivate then.When certain degree is arrived in cell proliferation, can be by reducing the temperature to below the LCST, make hydrogel surface hydrophilic, this like cell just breaks away from hydrogel, thereby obtain whole cell sheet, avoided the infringement of the utilization pair cell that enzymic digestion brought, gained cell sheet can be used for transplanting in the body reconstruction of three-dimensional tissue.
The synthetic method of PNIPAAm hydrogel mainly contains chemical polymerization and radio polymerization.Chemical polymerization PIPAAm hydrogel method is very ripe, and much more document has report.The temperature response PIPAAm hydrogel of chemical synthesis process preparation has limited its application in the cell sheet engineering preparation because of having following shortcoming: (1) chemical synthesis process, residue is more, for follow-up cell cultures certain toxicity is arranged.(2) chemical synthesis process can not be grafted to the PIPAAm hydrogel on the polystyrene type cultivation vessel, therefore, can not make culturing cell adherent in the PIPAAm hydrogel surface, more can not obtain the cell sheet.Radio polymerization is that the polyreaction of utilizing ionizing rays can cause organic monomer obtains macromolecular compound.Radio polymerization is be the mode that causes different with the main difference of the polyreaction (being chemical polymerization) that initiator causes, and chain reaction is once beginning, and company subsequently increases and what difference chain termination has not just had with usual way.Radio polymerization can be grafted to PNIPAAm in the common polystyrene culture dish or culture plate when carrying out the PNIPAAm monomer polymerization, gets final product culturing cell with this culture dish or culture plate.Radio polymerization mainly is divided into the polymerization that radionuclide, uv irradiating and rumbatron cause again by source of radiation.Than other two kinds of radiation modes, the electron beam of rumbatron is directed, also have dose rate height, power is big, radiated time is short, treatment capacity is big and the electron beam penetration power is low characteristics, be particularly suitable for making the PIPAAm temperature response hydrogel culture dish of thin layer.But, during simple rumbatron polymerization PIPAAm hydrogel, when total radiation dosage is lower than 240kGy, be not easy to form network-like polymkeric substance, only form linear polymkeric substance usually, soak the back linear polymer with ionized water and can dissolve (Fig. 1).Fourier's infared spectrum is found, the image that does not add the PIPAAm that produces behind the linking agent rumbatron radio polymerization is consistent with the image of linear PIPAAm, prompting does not add the PIPAAm that produces behind the linking agent rumbatron radio polymerization and only shows linear PIPAAm, fails to form network-like crosslinked PIPAAm hydrogel (Fig. 2).If once adopt the crosslinked PIPAAm hydrogel of the rumbatron of the above dosage of 200kGy, though can obtain network-like polymkeric substance, the electronics of high dosage quickens to follow the high temperature of moment, causes the cultivation vessel distortion impaired (Fig. 3) of polystyrene type usually.
Summary of the invention
At above-mentioned problems of the prior art, purpose of the present invention at first is to provide a kind of cell sheet engineering that obtains of cultivating with the PIPAAm hydrogel.
Another object of the present invention provides the preparation method of above-mentioned cell sheet engineering.
Above-mentioned purpose realizes by following technical scheme:
A kind of preparation method of cell sheet engineering specifically may further comprise the steps:
(1) containing mass concentration in the covering of cell cultures support surface is 0.1%～1.5% linking agent N, the N-N-isopropylacrylamide monomer solution of N '-methylene-bisacrylamide; The monomeric mass concentration of described N-N-isopropylacrylamide is 40%～60%.
(2) step (1) gained solution is carried out the rumbatron radiation synthesis, total radiation dosage is 240kGy～300kGy, divides three～six radiation synthetic continuously, and each radiation quantity is lower than 200kGy.Obtained covering the cell cultures upholder of poly N-isopropyl acrylamide hydrogel;
(3) the cell cultures upholder that step (2) is obtained is put in 4 ℃ the deionized water, soaked 5～8 days, during change water 4～7 times, after drying and sterilizing, seal standby;
(4) to make the poly N-isopropyl acrylamide hydrogel be hydrophobic phase to controlled temperature, seeds cells in the cell cultures upholder that step (3) obtains culturing cell; Change temperature, making the poly N-isopropyl acrylamide hydrogel is aqueous favoring, by polymeric membrane absorption or clamp forceps clamping method, isolates cell sheet engineering.
The described N-N-isopropylacrylamide of step (1) monomer preferably uses the monomer through recrystallization.It is that 1: 1 benzene and Virahol carries out that monomeric recrystallization preferably adopts volume ratio.
The described N-N-isopropylacrylamide of step (1) monomer solution preferably adopts Virahol as solvent.Water is during as the NIPAAm solvent monomer, and solution can not evenly stretch and be layered on the cultivation vessel surface, may be polar solvent with water then, and that Virahol is a non-polar solvent is relevant.In addition, water is also inhomogeneous as the crosslinking by ionizing radiation effect that the NIPAAm solvent monomer carries out, and some partial result is good, and some position can not be crosslinked fully.Therefore, be that the NIPAAm solvent monomer is better with the Virahol.
The N-N-isopropylacrylamide monomer overlay capacity of the described cell cultures support surface of step (1) is preferably 3mg～5mg/cm
2
The add-on of the described linking agent of step (1) is preferably 10 μ g～30 μ g/cm
2
The described cell cultures upholder of step (1) is preferably polystyrene board or tetrafluoroethylene material, polystyrene culture dish (tissue culture polystyrene for example, TCPS) or on six orifice plates, perhaps MILLICELL-CM insert culture dish (diameter: 30mm, aperture: 0.4 μ m, tetrafluoroethylene (Polytetrafluoroethylene, PTFE) filter membrane material).
Preferably, vacuum tightness≤2.2 * 10 of the described rumbatron of step (2)
-6KPa.
Ethylene oxide sterilizing is preferably adopted in the described sterilization of step (3).
Utilize phase transition temperature that cell sheet engineering is separated preferred following scheme in the step (4):
Inoculating cell is to the cultivation vessel of grafting PIPAAm hydrogel; Put 5%CO
237 ℃ of cultivations of incubator when culturing cell reaches 80～90% fusions, are transferred to 20 ℃ incubator with cultivating vessel, cultivate after 1 hour, the hydrogel surface that cell emersion in blocks thickens in swelling by polymeric membrane absorption or clamp forceps clamping method, is isolated DNAcarrier free cell sheet engineering.
The inoculation of above-mentioned cell is suitable for the inoculation method of cell cultures routine, for example: get the cell that is in exponential phase of growth in the cultivation, draw nutrient solution, add 0.25% pancreatin-0.02%EDTA, be advisable to flood cell, put (5%CO in the incubator
2, 37 ℃) digested several minutes, when finding that attached cell becomes circle, add the substratum that contains serum immediately and stop digestion.Blow and beat into the individual cells suspension with suction pipe, centrifugal.Remove supernatant, add nutrient solution, blow and beat into single cell suspension,, be inoculated on the cultivation vessel of grafting PIPAAm hydrogel by required concentration.
The cell sheet engineering that the present invention also provides aforesaid method to obtain certainly, owing to kept natural ECM composition from the isolated biotechnology cell of PIPAAm hydrogel surface sheet, these ECM compositions have the effect of tamanori, therefore, can easily these cell sheets be bonding on the histoorgan that need carry out transplantation treatment, need not to sew up.
The insert culture dish of grafting PIPAAm hydrogel can carry out the cultivation of cell sheet equally, method is the same, in addition, can carry out bottom culture dish cultivation and raise cell, the insert culture dish of PIPAAm hydrogel makes up the cell sheet, by the better cell sheet of method acquisition vigor of co-cultured cell; The insert culture dish of PIPAAm hydrogel also can pass through the solution-air culture technique, obtains to have the epithelial cell sheet of branch confluent monolayer cells.
The present invention also provides a kind of multi-layer cellular sheet, and this multi-layer cellular sheet carries out multilayer according to a conventional method with above-mentioned organizational project cell to be cultivated and to obtain, and perhaps vertically integrates by adhesion protein liquid to obtain;
Described adhesion protein liquid comprises in the following protein at least a:
10 μ g～100 μ g/ml lns, 5 μ g～50 μ g/ml fibronectins, 100 μ g～1mg/ml peptide arginyl-glycyl-aspartoyl-Serine tetrapeptide (Arg-Gly-Asp-Ser, RGDS), 100mg～300mg/ml Fibrinogen, 10 μ g～100 μ g/mL type i collagens, 10 μ g～100 μ g/ml IV Collagen Type VIs;
Described collagen dilutes with 0.006～0.1mol/L acetate, other albumen NaHCO of pH 8.3
3Solution dilution;
After described Fibrinogen adds, need to add the zymoplasm 200～1000U/ml of equivalent and 10% liquid calcium chloride of equivalent.
With respect to prior art, the present invention has following beneficial effect:
1, the synthetic employing of PNIPAm hydrogel in conjunction with chemically crosslinked and and rumbatron radiating method, overcome the shortcoming when two kinds of methods are used separately.Both obtained network-like PIPAAm aquogel polymer and with the cultivation vessel grafted effect of polystyrene type, it is impaired not occur cultivating the vessel distortion again, the linking agent that adds minute quantity, after synthesizing polymkeric substance, pass through immersion treatment, few or the nothing of residue, growth is avirulent substantially for cell.
2, the cell sheet of temperature response PIPAAm hydrogel making can keep cell and intercellular desmosome structure, ZO-1 tight connecting device and other iuntercellular syndeton, can keep basilar membrane albumen and with bind relevant many compositions as integrating element etc., complete cell sheet engineering also has certain intensity.
3, the multi-layer cellular sheet all is by a plurality of unicellular overlapping placements at present, rely on the attachment proteins matter bonding of cell sheet self to be integrated into the multi-layer cellular sheet, but, in some cases, the multi-layer cellular sheet possibility bondability that this overlapping placement is integrated is not enough, be easy to loose, can remedy such and insufficient and use adhesion protein liquid.
Description of drawings
When Fig. 1 has shown rumbatron polymerization PIPAAm hydrogel, when total radiation dosage is lower than 240kGy, be not easy to form network-like polymkeric substance, only form linear polymkeric substance usually, soaking the back linear polymer with ionized water can dissolve.
Fig. 2 finds for PIPAAm hydrogel Fourier infared spectrum: the image (below lines) consistent with the image of linear PIPAAm (top lines) that does not add the PIPAAm that produces behind the linking agent rumbatron radio polymerization.
When Fig. 3 has shown rumbatron polymerization PIPAAm hydrogel, once adopt the electronics of the above dosage of 200kGy to quicken to follow the high temperature of moment, cause that usually the cultivation vessel distortion of polystyrene type is impaired, the NIPAAm monomer is not seen polymerization, and it is flower-shaped to become rice krispies.
Fig. 4 has shown the form of cultivation vessel when temperature is higher than LCST of grafting PIPAAm hydrogel.
Fig. 5 has shown the form of cultivation vessel when temperature is lower than LCST of grafting PIPAAm hydrogel.
Fig. 6 is the Fourier's infared spectrum that adds the PIPAAm that produces behind the linking agent rumbatron radio polymerization.
Fig. 7 is a chemical process synthetic PIPAAm hydrogel Fourier infared spectrum.
Fig. 8 is Fourier's infared spectrum of PIPAAm hydrogel: wherein, and the PIPAAm hydrogel that upper curve produces after representing the NIPAAm monomer through the rumbatron radio polymerization; The PIPAAm hydrogel that on behalf of chemically crosslinked, lower curve produce.
Fig. 9 is a PIPAAm hydrogel atomic force microscope 2D image.
Figure 10 is a PIPAAm hydrogel atomic force microscope 3D rendering.The atomic force microscope inspection finds, PIPAAm hydrogel surface pattern has slight coarse.
Figure 11 is a PIPAAm hydrogel scanning electron microscope image, and the PIPAAm hydrogel surface is fine and close level and smooth, does not see obvious gauffer, and hydrogel edges is concavo-convex coarse more obvious.
Figure 12 is that NIPAAm monomer overlay capacity is greater than 5mg/cm
2The time the flower-shaped form of rice krispies that forms.
Figure 13 is the cultivation vessel of stand-by grafting PIPAAm hydrogel behind the sterilization in the ethylene oxide sterilizer.
Figure 14 is that the enzymic digestion tissue block is carried out the image under the 5th day the inverted microscope of corneal epithelial cell that cellular segregation cultivates, and the visible tissue piece is residual.
Figure 15 is the image under 9 days the inverted microscope of corneal epithelial cell cultivation, as seen merges corneal epithelial cell in blocks and marginal texture thereof.
Figure 16 is the oral mucosa epithelial cell in PIPAAm hydrogel surface adherent growth.
Figure 17 is the keratocyte in PIPAAm hydrogel surface adherent growth.
Figure 18 is the keratocyte in PIPAAm hydrogel surface edge adherent growth, shows neat keratocyte sheet marginal texture.
Figure 19 is the DNAcarrier free H﹠amp that contains the monolithic keratocyte sheet of multiple confluent monolayer cells; The painted tissue slice image of E.
Figure 20 is the scanning electron microscope image of monolithic keratocyte sheet, demonstrates cell and arranges surface tissue closely.
Figure 21 is the scanning electron microscope image of monolithic keratocyte sheet, demonstrates the marginal texture of keratocyte sheet.
Figure 22 is the 3T3 cell in PIPAAm hydrogel surface adherent growth.
Figure 23 is the H﹠amp of DNAcarrier free two composite cornea stroma cell sheets; The painted tissue slice image of E.
Figure 24 is the scanning electron microscope image of multi-disc composite cornea stroma cell sheet, demonstrates cell and arranges surface tissue closely.
Figure 25 is that DNAcarrier free multi-disc composite cornea stroma cell sheet forms early stage H﹠amp; The painted tissue slice image of E, bonding undertighten between each cell sheet.
Figure 26 is the H﹠amp that DNAcarrier free multi-disc composite cornea stroma cell sheet forms; The painted tissue slice image of E, bonding tight between each cell sheet, form complete 7-10 layer keratocyte sheet.
Embodiment
Below in conjunction with embodiment, the present invention is done detailed description further, but implementation of the present invention is not limited thereto.
The structure of embodiment 1 corneal epithelial cell sheet
1, the preparation of PNIPAm hydrogel grafting culture dish
(1) recrystallization monomer
(the utilization volume ratio is that 1: 1 benzene and Virahol carries out recrystallization, and is stand-by behind the NIPAAm monomer drying behind the recrystallization for N-isopropylacrylamide, NIPAAm) (the Shanghai thing is competed Science and Technology Ltd.) with buying the N-N-isopropylacrylamide monomer that comes.
(2) preparation monomer solution
The preparation mass concentration is 40% NIPAAm monomer solution, and containing mass concentration is 0.1% linking agent N, and (N, N-methylene bisacrylamide MBA), more than are by the preparation of solution Virahol N '-methylene-bisacrylamide, and be stand-by behind the concussion mixing.
(3) radiation synthesis monomer solution
Get the above-mentioned NIPAAm monomer solution that has prepared, (tissue culturepolystyrene TCPS) on the culture dish, evenly stretches by the tension force of monomer solution self and to be layered on the cultivation vessel surface to join the polystyrene board of 35mm.Monomer is 4mg/cm at the overlay capacity of cultivating vessel surface
2Put it to rumbatron (dynamitron, Chinese Academy of Sciences's Shanghai nuclear research manufacturing) it is synthetic to carry out electron radiation on the dolly, data during the rumbatron radiation synthesis are: radiation dose is 240kgy, divide three radiation synthetic continuously with dolly, rumbatron vacuum tightness is 2.2 * 10
-6KPa.Through the rumbatron radiation effect, making the NIPAAm monomer polymerization is the PIPAAm hydrogel, meanwhile, and NIPAAm monomer and the graft polymerization of cultivation vessel.
Present embodiment grafted PIPAAm hydrogel has the temperature response characteristics: when temperature was higher than LCST, volumetric shrinkage became oyster white thin layer (Fig. 4), and when temperature was lower than LCST, volume swelling became transparent hydrogel (Fig. 5).PIPAAm hydrogel Fourier infared spectrum finds, near the bands of a spectrum that occur 1644cm-1 are that the carboxyl stretching vibration by acid amides produces, i.e. acid amides I band; And be acid amides II band at the bands of a spectrum at 1538cm-1 place, be that combination by N-H flexural vibration in the amide group and C-H stretching vibration absorbs and produces.In addition, absorption peak has all appearred in wave number near 1386cm-1 and 1367cm-1, and this is sec.-propyl-CH (CH of NIPAAm
3)
2In the characteristic spectrum of symmetrical C-H vibration.PIPAAm hydrogel Fourier infared spectrum image after the adding little amount of crosslinking agent is consistent with the main component image of chemosynthesis, close (Fig. 6 of PIPAAm hydrogel main component that PIPAAm hydrogel component that produces behind the prompting NIPAAm monomer process rumbatron radio polymerization and chemically crosslinked produce, Fig. 7, Fig. 8).Autoprobe CP Research type atomic force microscope (U.S. THERMO company) check to find, PIPAAm hydrogel surface pattern have slight coarse (Fig. 9, Figure 10).JEOLT-300SEM scanning electronic microscope (Jeol Ltd.) checks further and confirms that the PIPAAm hydrogel surface is fine and close level and smooth, does not see obvious gauffer, hydrogel edges concavo-convex coarse obviously (Figure 11).Monomer is 3mg～5mg/cm at the overlay capacity of cultivating vessel surface
2, overlay capacity is less than 3mg/cm
2The time, can not radio polymerization be the PIPAAm hydrogel of slabbing; Overlay capacity is greater than 5mg/cm
2The time, form the rice krispies style easily, can not form the PIPAAm hydrogel (Figure 12) of thin layer.In addition, culturing cell also is difficult to attach thereon.The growth that culturing cell is attached to the PIPAAm hydrogel surface is that to be adsorbed in hydrogel relevant with the adhesion protein (for example fibronectin) of cell, if the monomer overlay capacity is too big, form the PIPAAm hydrogel of thick-layer, be difficult to adsorbing fiber and connect compositions such as albumen.The MBA add-on is generally 10 μ g～30 μ g/cm
2, MBA is very few less crosslinked or only form linear crosslinkedly, and the suction back is loose; The excessive degree of crosslinking of MBA is too big, loses water absorption character.
Water is during as the NIPAAm solvent monomer, and solution can not evenly stretch and be layered on the cultivation vessel surface, may be polar solvent with water then, and that Virahol is a non-polar solvent is relevant.In addition, water is also inhomogeneous as the crosslinking by ionizing radiation effect that the NIPAAm solvent monomer carries out, and some partial result is good, and some position can not be crosslinked fully.Therefore, be that the NIPAAm solvent monomer is better with the Virahol.
(4) processing of the PIPAAm hydrogel behind the radiation synthesis
Temperature-sensitive hydrogel behind the radiation synthesis is put in 4 ℃ the deionized water, soaked 5 days, during change water 4 times, put into 50 ℃ of baking boxs and dry.The cultivation vessel of grafting PIPAAm hydrogel seal paper-plastic package by sealing machine, put an ethylene oxide sterilizing indication test paper before sealing, put it to sterilization in the HM-23 ethylene oxide sterilizer (Shenyang De Long Electronics Co., Ltd.), when test paper becomes blueness by redness, the expression sterilization is up to standard, it is stand-by to take out the back, seals off use (Figure 13) when carrying out cell cultures again.
2, cell cultures on the hydrogel and transplanting
(1) nutrient solution
Nutrient solution: A. adds epithelial cell nutrient solution (the supplemental hormonal epithelial medium of hormone, SHEM): DMEM/F12 (3: 1), 10 μ g/L Urogastrons, 1000U/L Regular Insulin, 0.1mg/L Toxins,exo-, cholera, 0.18mM/L VITAMIN B4,0.5mg/L hydrocortisone, 1.2g/L NaHCO3,10% foetal calf serum (FBS), 100,000 U/L penicillin and 100mg/L Streptomycin sulphate.B. low calcium keratinocyte substratum (keratinocyte serum freemedium, KSFM), Ca
2+Concentration be 0.07mM～0.09mM.Cultivate reagent except that specifying, adopt U.S. Gibco company product.Other chemical reagent adopt U.S. Sigma company product except that except that specifying.
(3) draw materials
Under sterilising conditions, use the utensil of sterilization to carry out relating operation as much as possible.In-vitro eyeball is drawn materials: separate the corneal limbus annular tissue of wide about 2mm, it is on average cut is 8.Draw materials from body live body eyeball: about 2 * 3 * 1mm of shallow-layer isolated cornea edge
3
(4) enzymic digestion tissue block carry out cellular segregation
Organize fritter to put in the corneal epithelial cell nutrient solution that 4 ℃ of serum-frees contain 1.2U/ml neutral protease and 100mM sorbyl alcohol above-mentioned corneal limbus, about 10 hours, under the dissecting microscope, loosening epithelial lining is separated with micro-tweezers.Place the 15ml centrifuge tube, add about 5ml trypsin solution (0.05% trypsinase and 0.02%EDTA) in the centrifuge tube, 37 ℃ digested 30 minutes down, and it is centrifugal to stop the rearmounted 1000rpm/min of digestion, 5 minutes.Siphon away supernatant liquor after centrifugal, add 10% foetal calf serum, the piping and druming of DMEM/F12 nutrient solution is made cell suspension, with 2 * 10
4/ ml cell count inoculation, carry out cell former be commissioned to train foster.DM/L inverted biologic microscope (Leca company) check down find the enzymic digestion tissue block carry out the corneal epithelium that cellular segregation cultivates and merge corneal epithelial cell in blocks (Figure 14, Figure 15).With 1 * 10
5/ ml cell density is inoculated in the cultivation vessel and carries out the passage cell cultivation, uses the passage cell of cultivating in 5 generations.
(5) the corneal epithelial cell sheet of multiple confluent monolayer cells
With the corneal limbal epithelial cell cultivated with 2 * 10
4The cell density of/ml is inoculated in the described culture dish surface that step 1 obtains, and carries out single cell culture.
(6) separation of corneal epithelial cell sheet is obtained
Adopt nitrocellulose membrane adherent cell sheet directly to separate.
(7) the corneal epithelial cell sheet is transplanted
Transplant object: (1) laboratory animal: to New Zealand white rabbit or other animal, 4mm place outside the distance corneal limbus, remove corneal epithelium and the conjunctival epithelium of about 0.1～1mm with the curved scissors cutter, make the surperficial stem cell deficiency disease animal model of eye, remove fully with fluorescein eye table dyeing affirmation corneal epithelial cell.After 4 weeks, this animal eye surface is covered by remaining on every side conjunctival epithelium, anterior corneal surface muddiness and cornea rebirth blood vessel occur.(2) clinical patients: the scar tissue that comprises the chemical injury delay corneal epithelial defect patient of acute phase, chemical injury chronic phase is invaded patients such as cornea pathology, ocular pemphigoid such as the patient that causes the cornea rebirth blood vessel and the corneal opacity, eye surface diseases that limbal stem cell lacks, corneal limbus dysfunction.That transplants can be used for the treatment of various keratopathy without any the corneal epithelial cell sheet of biomaterial, so just is expected to well solve the various after-operation response that occur because of the effect of the degraded of biomaterial or in-vivo tissue and biomaterial.Employing is cultivated from the healthy corneal epithelial cell of body can obtain epithelium of autologous cornea cell sheet, it is transplanted can solve the insufficient contradiction of cornea donor and remove the postoperative rejection that allosome corneal epithelial cell sheet is transplanted appearance.
Operation method: the full shallow-layer flaggy of pathology cornea is separated excision, remove the anterior corneal surface tissue of muddy, conjunctival epitheliumization and neovascularization, fully stop blooding and clean, expose transparent corneal stroma, produce the acceptor plant bed.Skin graft on above-mentioned and the adherent functionally active artificial cornea of nitrocellulose membrane is contacted attaching with plant bed, bind, reach the cell sheet and transplant, transplant the back and remove nitrocellulose membrane by the tissue between cell sheet adhesion protein and the corneal stroma.The cell sheet that can use the protection of bandage type contact lens to transplant in case of necessity.Postoperative, local use microbiotic eye drop and eye ointment, 2 times/day.After in the corneal epithelial cell sheet transplant, whether the corneal epithelial cell sheet of using fluorescent staining inspection transplanting has the barrier function, confirms the transplantation effect of epithelial cell sheet.
The structure of above-mentioned corneal epithelial cell sheet and the seed cell of transplanting also can be used oral mucosa epithelial cell (Figure 16) and other cell except using corneal limbal epithelial cell.When the autologous corneal limbus epithelial cell that is difficult to secure good health, can use from the oral mucosa epithelial cell of body and other cell seed cell as an alternative, have equally and solve the insufficient contradiction of cornea donor and reduce the advantage that allosome corneal epithelial cell sheet is transplanted the postoperative rejection that occurs.
The structure of embodiment 2 corneal epithelial cell sheets
1, the preparation of PNIPAm hydrogel grafting culture dish
(1) recrystallization monomer
With embodiment 1.
(2) preparation monomer solution
The preparation mass concentration is 60% NIPAAm monomer solution, and containing mass concentration is 0.4% linking agent N, and (N, N-methylene bisacrylamide MBA), more than are by the preparation of solution Virahol N '-methylene-bisacrylamide, and be stand-by behind the concussion mixing.
(3) radiation synthesis monomer solution
Get the above-mentioned NIPAAm monomer solution that has prepared, join MILLICELL-CM insert culture dish [diameter: 30mm, aperture: 0.4 μ m, tetrafluoroethylene (Polytetrafluoroethylene, PTFE) (Millipore company, the U.S.) filter membrane material] on the surface, evenly stretch by the tension force of monomer solution self and to be layered on the cultivation vessel surface.Monomer is 4.5mg/cm at the overlay capacity of cultivating vessel surface
2Put it to that to carry out electron radiation on the dolly of rumbatron synthetic, the data during the rumbatron radiation synthesis are: radiation dose is 300kgy, divides six radiation synthetic continuously with dolly, and rumbatron vacuum tightness is 2.2 * 10
-6KPa.Through the rumbatron radiation effect, making the NIPAAm monomer polymerization is the PIPAAm hydrogel, meanwhile, and NIPAAm monomer and the graft polymerization of cultivation vessel.
The hydrogel that grafted PIPAAm hydrogel that obtains and embodiment obtain has similar characteristics.
(4) processing of the PIPAAm hydrogel behind the radiation synthesis
Temperature-sensitive hydrogel behind the radiation synthesis is put in 4 ℃ the deionized water, soaked 8 days, during change water 7 times, put into 50 ℃ of baking boxs and dry.After this processing is with embodiment 1.
2, cell cultures on the hydrogel and transplanting
(1) nutrient solution
With embodiment 1.
(3) draw materials
With embodiment 1.
(4) enzymic digestion tissue block carry out cellular segregation
Organize fritter to put in the corneal epithelial cell nutrient solution that 4 ℃ of serum-frees contain 2.4U/ml neutral protease and 100mM sorbyl alcohol above-mentioned corneal limbus, about 18 hours, under the dissecting microscope, loosening epithelial lining is separated with micro-tweezers.Place the 15ml centrifuge tube, add about 5ml trypsin solution (0.05% trypsinase and 0.02%EDTA) in the centrifuge tube, 37 ℃ digested 30 minutes down, and it is centrifugal to stop the rearmounted 1000rpm/min of digestion, 5 minutes.Siphon away supernatant liquor after centrifugal, add 10% foetal calf serum, the piping and druming of DMEM/F12 nutrient solution is made cell suspension, with 1 * 10
6/ ml cell count inoculation, carry out cell former be commissioned to train foster.With 1 * 10
6/ ml cell density is inoculated in the cultivation vessel and carries out the passage cell cultivation, uses the passage cell of cultivating in 5 generations.
(5) the corneal epithelial cell sheet of multiple confluent monolayer cells
With the corneal limbal epithelial cell cultivated with 1 * 10
6The cell density of/ml is inoculated in the insert culture dish surface that step 1 obtains, carry out co-culture of cells: will cultivate the 3T3 inoblast of fusion or the ametycin of human embryonic fibroblast and 4 μ g/mL in advance and hatch altogether 2 hours, PBS washing 2 times is after the trysinization, with 1 * 10
4/ cm
2In inoculation culture ware or the culture plate.Just this culture dish or culture plate in put into the insert culture dish that contains corneal limbal epithelial cell thereafter.Put into 5% CO
237 ℃ of cultivations in the incubator, 97% humidity, changed liquid 1 time in per 2～3 days, the conventional cultivation after 7～10 days carried out gas-liquid and cultivated, and promptly reduces the nutrient solution add-on, add the nutrient solution plane and only reach insert culture dish surface, make the more ingresss of air of surface epithelial cell of cultivation, add weekly with what mitomycin was handled and raise cell, gas-liquid is cultivated and is obtained multiple layer corneal epithelial cell sheet after 7～10 days.
(6) separation of corneal epithelial cell sheet is obtained
The method of obtaining multiple layer corneal epithelial sheet is: after the cultivation of above-mentioned multiple layer corneal epithelial cell sheet is finished, reduce nutrient solution in the insert culture dish, on culturing cell, cover nitrocellulose membrane, putting into incubator cultivated 30 minutes for 20 ℃, under temperature condition below 20 ℃, the PIPAAm from hydrophilic loose ball of string structure peels off with the nitrocellulose membrane that binds whole cell sheet.
(7) the corneal epithelial cell sheet is transplanted
The available method identical with embodiment transplanted the cell sheet.
Embodiment 3 endothelial cell sheet for cornea
Carry out seed cell by the conventional cultural method of endothelial cell and cultivate, adopt embodiment 1 or 2 described corneal epithelial cell sheets to make up similar method and make up endothelial cell sheet for cornea.Endothelial cell sheet for cornea is transplanted and can be treated the endothelial cell pathology and inner skin cell function loses compensatory grade for patient: remove the corneal posterior lamella and the corneal endothelial layer of pathology, divest operation method such as corneal endothelium transplanting and carry out the endothelial cell sheet for cornea transplanting by being similar in penetrating keratoplasty, the dark lamellar cornea skin grafting dermepenthesis and descemet's membrane.Endothelial cell sheet for cornea is transplanted does not need suture to sew up usually, adopts the tissue between cell sheet adhesion protein and the corneal posterior lamella to bind, and reaches the cell sheet and transplants.Or adopt above-mentioned at least a adhesion protein liquid that the cell sheet is attached to corneal posterior lamella.Slit lamp observation corneal transparency situation checks corneal edema whether occurs, confirms the endothelial cell sheet for cornea transplantation effect.The structure of above-mentioned endothelial cell sheet for cornea and the seed cell of transplanting also can be used other cell except using endothelial cell.
Embodiment 4 keratocyte sheets
Carrying out seed cell by the conventional cultural method of keratocyte cultivates, adopt above-mentioned corneal epithelial cell sheet to make up similar method and make up keratocyte sheet (Figure 17, Figure 18)), reduce the temperature separation by aforesaid method and can obtain DNAcarrier free keratocyte sheet.The H﹠amp of keratocyte sheet; The painted tissue slice of E is learned in opticmicroscope undertissue and is checked discovery, and carrier free monolithic keratocyte sheet inner cell is arranged closely, contains the multiple confluent monolayer cells structure (Figure 19) of layer 2-3.Inspection discovery monolithic keratocyte sheet cell is arranged surface tissue (Figure 20) and marginal texture (Figure 21) closely under the scanning electronic microscope.The keratocyte sheet is transplanted can treat the keratopathy patient: remove the corneal stroma pathological tissues, carry out the transplanting of keratocyte sheet by being similar to lamellar cornea transplantation method, the keratocyte sheet is transplanted does not need suture to sew up usually, adopt the tissue between cell sheet adhesion protein and the cornea to bind, reach the cell sheet and transplant.Or adopt above-mentioned at least a adhesion protein liquid that the cell sheet is attached to cornea tissue.Slit lamp observation cornea situation is confirmed keratocyte sheet transplantation effect.The structure of above-mentioned keratocyte sheet and the seed cell of transplanting also can be used other cell (Figure 22) except using keratocyte.
The preparation of embodiment 5 multi-disc composite cornea stroma cell sheets
Carry out seed cell by the conventional cultural method of keratocyte and cultivate, adopt above-mentioned corneal epithelial cell sheet to make up similar method and make up monolithic keratocyte sheet, adopt above-mentioned cell sheet to separate the method for obtaining and obtain a plurality of monolithic keratocyte sheets.Integrate the multi-disc cultural method according to the overlapping placement of following monolithic and obtain multi-disc composite cornea stroma cell sheet: after adopting above-mentioned keratocyte sheet to make up similar method structure keratocyte sheet, the nutrient solution that reduces the cultivation vessel is to the bottom of cultivating vessel, above-mentioned a plurality of monolithic keratocyte sheets longitudinal overlap piecewise placement is integrated into the multi-layer cellular sheet, prevent that the cell sheet from curling and disengaging, leave standstill several minutes to a few hours, add enough nutrient solutions, routine is cultivated a few hours to a few days.Adopt above-mentioned cell sheet to separate the method for obtaining and obtain a multi-disc composite cornea stroma cell sheet, produce transplantable two (Figure 23) or multi-disc composite cornea stroma cell sheet (Figure 24).Multi-disc composite cornea stroma cell sheet forms in early days, and bonding undertighten (Figure 25) between each cell sheet is bonding tight between each cell sheet after the cultivation of placement a few hours to a few days, can form complete 7-10 layer keratocyte sheet (Figure 26).Owing to kept natural ECM protein ingredient from the isolated monolithic keratocyte of PIPAAm hydrogel surface sheet, these ECM albumen have the effect of tamanori, therefore, the overlapping placement of monolithic is integrated the multi-disc cultural method and can be obtained closely complete multi-disc composite cornea stroma cell sheet of cellularstructure.The implantation method of multi-disc composite cornea stroma cell sheet is with embodiment 4.
The multi-disc composite cornea cell sheet that embodiment 6 various kinds of cell sheets are vertically integrated
Obtain the multi-disc composite cornea cell sheet that the various kinds of cell sheet is vertically integrated with at least two kinds in above-mentioned three kinds of corneal cell sheets (corneal epithelial cell sheet, endothelial cell sheet for cornea and keratocyte sheet) by following three kinds of methods:
Method one is that the multi-disc culture method is integrated in the overlapping placement of monolithic: the multi-disc cultural method is integrated in the overlapping placement of monolithic of the preparation of the multi-disc composite cornea stroma cell sheet of employing embodiment 5, utilizes ECM albumen adhesive effect natural between the cell sheet to obtain the multi-disc composite cornea cell sheet that the various kinds of cell sheet is vertically integrated.
Method two is that adhesion protein liquid sticks method: after adopting above-mentioned cell sheet to make up three kinds of corneal cell sheets of similar method structure, the nutrient solution that reduces the cultivation vessel is to the bottom of cultivating vessel, adopt above-mentioned adhesion protein liquid, one or more uses of adhesion protein liquid, above-mentioned three kinds of corneal cell sheet monolithics are vertically sticked piecewise, prevent that the cell sheet from curling and disengaging, leave standstill several minutes to a few hours, add enough nutrient solutions, routine is cultivated a few hours to a few days.Adopt above-mentioned cell sheet to separate the method for obtaining and obtain the multi-disc composite cornea cell sheet that a various kinds of cell sheet is vertically integrated.
Method three is that above-mentioned two kinds of methods are mixed application: it is to rely on the attachment proteins matter bonding of cell sheet self to be integrated into multi-disc composite cornea cell sheet that the multi-disc culture method is integrated in the overlapping placement of monolithic, but, in some cases, the multi-disc compound cells sheet possibility bondability that this overlapping placement is integrated is not enough, be easy to loose, can remedy such and insufficient and mix to use adhesion protein liquid.
The multi-disc composite cornea cell sheet that above-mentioned various kinds of cell sheet is vertically integrated comprises: flaggy corneal transplantation before can carrying out after corneal epithelial cell sheet (monolithic) is vertically integrated with keratocyte sheet (multi-disc); Endothelial cell sheet for cornea (monolithic) can carry out the metaplax layer corneal transplantation after vertically integrating with keratocyte sheet (multi-disc); Corneal epithelial cell sheet (monolithic) can carry out the holostrome corneal transplantation with keratocyte sheet (multi-disc) and the vertical integration of endothelial cell sheet for cornea (monolithic).Above-mentioned corneal transplantation can be treated the relevant diseases of cornea different sites.
Embodiment 7 retinal pigment epithelium sheets
Enzymic digestion or tissue block adherent method carry out retinal pigment epithelium separation and former be commissioned to train foster, the conventional retinal pigment epithelium of cultivating, make up similar method according to above-mentioned corneal epithelial cell sheet and make up the retinal pigment epithelium sheet, the retinal pigment epithelium sheet is transplanted in subretinal space, can be treated because the fundus oculi disease that the retinal pigment epithelium metabolic disturbance causes.The transplanting of retinal pigment epithelium sheet is better than retinal pigment epithelium suspension injection transplantation part and is can keep after the former transplants normal polarity under the retinal pigment epithelium physiological status, and correct polar contribution has great importance to the function of keeping retinal pigment epithelium.The structure of above-mentioned retinal pigment epithelium sheet and the seed cell of transplanting also can be used other pigment epithelial cell except using retinal pigment epithelium, iris epithelial cell.
Carry out seed cell by the conventional cultural method of retina neural sensory layer cell (photoreceptor cell, relay cell, ganglion cell) and cultivate, adopt above-mentioned corneal epithelial cell sheet to make up similar method and make up the optic cell sheet.Retina neural sensory layer cell sheet is transplanted can treat the retinopathy patient.The structure of above-mentioned retina neural sensory layer cell sheet and the seed cell of transplanting also can be used other cell except using retina precursor cell (retinal progenitor cells).
Embodiment 9 retina cell's sheets
Above-mentioned retinal pigment epithelium sheet and retina neural sensory layer cell sheet vertically integrated by unicellular overlapping placement or stick and vertically be integrated into compound retina cell's sheet by adhesion protein liquid, one or more uses of adhesion protein liquid, retina cell's sheet is transplanted can treat multiple retinopathy patient.
Embodiment 10 skin cells sheets
Similar with the structure of corneal cell sheet, both epidermis epithelial cell sheet can be made up respectively and the dermal cell sheet carries out transplantation treatment, also can be with epidermis epithelial cell sheet and dermal cell sheet by overlapping integration or stick and vertically be integrated into multi-disc composite skin cell sheet, transplant the treatment dermatosis again.
Embodiment 11 periodontal tissue's sheets
Separate the periodontium inoblast and carry out conventional cell cultures by enzymic digestion tissue block method, make up periodontium inoblast sheet by last method, transplant in periodontal, postoperative can be observed the regeneration of gum, regeneration gum and tooth adhere to closely, can treat gingival atrophy or other gum pathology.
Embodiment 12 myocardial cell's sheets
Make up myocardial cell's sheet by last method, a plurality of myocardial cell's sheets can overlapping placement vertically be integrated into the compound myocardial cell's sheet of multi-disc, also can vertically be integrated into multi-disc compound cells sheet by sticking.Can observe beating and bioelectrical activity of multi-disc compound cells sheet, multi-disc compound cells sheet can be transplanted to myocardium defect.
Can also make up cell sheets such as hepatic tissue, urinary system, respiratory system and carry out transplantation treatment by above-mentioned.
The foregoing description is a preferred implementation of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (9)
1. the preparation method of a cell sheet engineering is characterized in that specifically may further comprise the steps:
(1) containing mass concentration in the covering of cell cultures support surface is 0.1%～1.5% linking agent N, the N-N-isopropylacrylamide monomer solution of N '-methylene-bisacrylamide;
The monomeric mass concentration of described N-N-isopropylacrylamide is 40%～60%;
The N-N-isopropylacrylamide monomer overlay capacity of described cell cultures support surface is 3mg～5mg/cm
2
The add-on of described linking agent is 10 μ g～30 μ g/cm
2
(2) step (1) gained solution is carried out the rumbatron radiation synthesis, total radiation dosage is 240kGy～300kGy, divide three～six radiation synthetic continuously, each radiation quantity is lower than 200kGy, has obtained covering the cell cultures upholder of poly N-isopropyl acrylamide hydrogel;
(3) the cell cultures upholder that step (2) is obtained is put in 4 ℃ the deionized water, soaked 5～8 days, during change water 4～7 times, after drying and sterilizing, seal standby;
(4) to make the poly N-isopropyl acrylamide hydrogel be hydrophobic phase to controlled temperature, seeds cells in the cell cultures upholder that step (3) obtains culturing cell; Change temperature, making the poly N-isopropyl acrylamide hydrogel is aqueous favoring, by polymeric membrane absorption or clamp forceps clamping method, isolates cell sheet engineering.
2. the preparation method of cell sheet engineering according to claim 1 is characterized in that: the described N-N-isopropylacrylamide of step (1) monomer process recrystallization.
3. the preparation method of cell sheet engineering according to claim 2 is characterized in that: it is that 1: 1 benzene and Virahol carries out that described recrystallization is to use volume ratio.
4. the preparation method of cell sheet engineering according to claim 1, it is characterized in that: the described cell cultures upholder of step (1) is polystyrene or tetrafluoroethylene material.
5. the preparation method of cell sheet engineering according to claim 1 is characterized in that: vacuum tightness≤2.2 * 10 of the described rumbatron of step (2)
-6KPa.
6. the preparation method of cell sheet engineering according to claim 1, it is characterized in that: the described sterilization of step (3) is an ethylene oxide sterilizing.
7. the preparation method of cell sheet engineering according to claim 1 is characterized in that: in the step (4), specifically adopt following method to separate and obtain described cell sheet engineering:
Inoculating cell to grafting poly--the cultivation vessel of N N-isopropylacrylamide hydrogel on; Put 5%CO
237 ℃ of cultivations of incubator when culturing cell reaches 80～90% fusions, are transferred to 20 ℃ incubator with cultivating vessel, cultivated 1 hour, the hydrogel surface that cell emersion in blocks thickens in swelling by polymeric membrane absorption or clamp forceps clamping method, is isolated DNAcarrier free cell sheet engineering.
8. cell sheet engineering that each described method of claim 1-7 obtains.
9. a multiple stratification cell sheet engineering is characterized in that: be to be placed by the described cell sheet engineering longitudinal overlap of claim 8, obtain through the multiple stratification cultivation; Perhaps vertically integrate and obtain by adhesion protein liquid;
Described adhesion protein liquid comprises in the following protein at least a:
10 μ g～100 μ g/ml lns, 5 μ g～50 μ g/ml fibronectins, 100 μ g～1mg/ml arginyl-glycyl-aspartoyl-Serine tetrapeptide, 100mg～300mg/ml Fibrinogen, 10 μ g～100 μ g/mL type i collagens, 10 μ g～100 μ g/ml IV Collagen Type VIs;
Described collagen dilutes with 0.006～0.1mol/L acetate, other albumen NaHCO of pH 8.3
3Solution dilution;
After described Fibrinogen adds, need to add 200～1000U/ml zymoplasm of equivalent and 10% liquid calcium chloride of equivalent.
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| CN113088481A (en) * | 2012-08-24 | 2021-07-09 | 独立行政法人理化学研究所 | Method for producing retinal pigment epithelial cell sheet |
| CN103436444A (en) * | 2013-07-26 | 2013-12-11 | 上海瀚正生物技术服务有限公司 | Culture apparatus used for temperature sensitive cells, preparation method thereof and cell culturing method |
| CN104861177B (en) * | 2015-04-27 | 2017-10-10 | 苏州大学 | Hydrogel material and preparation method with fibrin ferment response thrombolysis ability |
| KR102469649B1 (en) * | 2016-12-22 | 2022-11-21 | 후지필름 가부시키가이샤 | cell culture substrate |
| CN111936620A (en) * | 2019-02-28 | 2020-11-13 | 京东方科技集团股份有限公司 | Retinal pigment epithelial cell membrane and preparation method thereof |
| CN110229783A (en) * | 2019-05-22 | 2019-09-13 | 中国人民解放军总医院 | A method of stem cell diaphragm is prepared based on warm ware |
| CN110448731A (en) * | 2019-07-23 | 2019-11-15 | 中国人民解放军总医院 | The method that temperature sensitivity culture dish prepares Autopsy Cases ball derived stem cells film property patch |
| CN114466922A (en) * | 2019-10-01 | 2022-05-10 | 国立大学法人大阪大学 | Method for producing fibrin sheet |
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