CN105254917B - A method of preparing cell patch using Sodium Alginate Hydrogel Films - Google Patents
A method of preparing cell patch using Sodium Alginate Hydrogel Films Download PDFInfo
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- CN105254917B CN105254917B CN201510735361.5A CN201510735361A CN105254917B CN 105254917 B CN105254917 B CN 105254917B CN 201510735361 A CN201510735361 A CN 201510735361A CN 105254917 B CN105254917 B CN 105254917B
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Abstract
The invention discloses one kind can be used for building cell sheets（cell sheet）Base material and the method for preparing cell patch using it.Diaphragm is made in sodium alginate first with the method for being freeze-dried and adding perforating agent, in order to introduce necessary reactive group, sodium alginate is needed by chemical modification, is modified to sodium alginate substrate surface or ontology using large biological molecule, you can is used for inoculating cell.Finally on base material after inoculating cell, pass through the regulation and control of culture environment, promote cell Proliferation and generates a large amount of extracellular matrixs, to form complete cell patch, it reuses certain density sodium citrate solution to dissolve base material sodium alginate, obtains the completely cell patch with good biological performance.The above-mentioned technology of the present invention have the characteristics that substrate it is dissolvable, suitable for a large amount of cells, easy to operate, at low cost, avoid conventional method influences caused by cell, and good biocompatibility.
Description
Technical field
The invention belongs to field of tissue engineering technology.More particularly, to a kind of cell membrane is prepared using Sodium Alginate Hydrogel Films
The method of piece.
Background technology
Building tissue engineering material tool, there are three elements, i.e. seed cell, timbering material, cell factor.Traditional group weaver
When the structure of journey material, the digestion of trypsase or proteolytic enzyme is usually required during obtaining seed cell, from
And cell suspension is obtained, but trypsase and proteolytic enzyme to a certain extent damage cell, can lead to cell
A large amount of losses and activity is low, this be also many cells with passage number increase form change and the reason of gradual aging it
One.Meanwhile cell and timbering material direct combination, cell utilization rate are low, it is difficult to form fine and close bone tissue.But also cell can be made
Na+/K+-ATP enzymes, glucose transporter enzyme -1（glucose transporter-1,GLUT-1）, sodium-glucose corotation
Carrier（sodium glucose cotransporter-1,SGLT-1）, aquaporin etc. is by different degrees of destruction
With damage, it is unfavorable for it and is adhered on nail and extracellular matrix secretion.In order to solve seed losing issue, researchers carry
Go out a kind of seed cell inoculation technique -- cell patch technology（Cell sheet technology, CST）.
Domestic and foreign scholars conduct in-depth research cell patch technology in terms of organizational project, the cell patch of preparation
As tissue engineering material, had the characteristics that compared with traditional material product：First, desired tissue engineering cell is met
The condition of holder；Meanwhile the cell sheets not only have suitable for cell Proliferation, differentiation and the three-D space structure of adherency；Also,
Have good constituent, have it is nontoxic, without side-effects, have good biocompatibility；Finally, it promotes to a certain extent
Into angiogenesis, promote migration and differentiation of host stem cells etc..The seed cell that application cell lamella technology obtains, both retained
Structure between cell and cell again remains the extracellular matrix of cell secretion（Extracelluar mix, ECM）, and
ECM is a kind of important component of cellular environment, and the various ECM ingredients of cell secretion form matrix interstitial and matrix membrane, because
For the tissue be suitble to cell growth thus can promote cell in vivo cell anchor on the framework.
Cell patch technology is proposed that he passes through this temperature sensitive with n-isopropyl acrylamide by Okano earliest
Surface is as cell sheets.At the same time, alkanethiol or small peptide are independently filled by surface, pass through electrochemical method point later
From, or a kind of enzyme of non-proteolytic enzyme is used, then the enzyme hydrolysis etc. is hydrolyzed by straightforward procedure.Temperature-reactive is thin
For born of the same parents' lamella technology using relatively broad, it utilizes temperature-reactive culture dish culture cell, under 37 DEG C of condition of culture, training
The surface for supporting basal part polymer is in hydrophobicity, and cell can be proliferated in culture dish bottom, but when temperature is reduced to critical-temperature 32
DEG C or less when, polymer surfaces become hydrophily from hydrophobicity, and form aquation between culture dish bottom and the cell of culture
Layer makes cell completely be detached from culture dish bottom in stratiform.
But change temperature makes base and cell after current CST mainly supports cell by thermally sensitive surface
Separate, or realized by the special albumen of surface modification, it is complicated there are technical operation the defects of, with other organizational projects
Method is the same, and when application cell diaphragm technology carries out tissue reconstruction, the acquisition of seed cell is relatively difficult；The cell sheets of superposition
When more than certain thickness, the necrosis of internal cell is often resulted in；Application cell diaphragm technology builds big functional organization still
It is a great problem that cell patch technology faces, limits its popularization and application.Therefore, explore it is a kind of without using enzyme solution and
The technology of acquisition cell patch simple to operate is particularly important.
Invention content
The technical problem to be solved by the present invention is to overcome the defects and deficiency of existing cell patch technology, explore a kind of utilization
The sodium alginate base material that surface is modified is the new method that substrate prepares cell patch, which not only has routine CST's
Advantage, it is important that its substrate can dissolve, and to cell without particular/special requirement, the features such as being suitable for a large amount of cells；At the same time, should
Technical operation is simple and convenient, at low cost, is not necessarily to large scale equipment.Cell sheets, the superposition of application cell lamella are prepared using the technology
The tissue of three-dimensional structure and the tissue of the method structure three-dimensional structure of cell sheets combination holder are built, with traditional group weaver
Cheng Xue methods combine the development for inherently promoting organizational engineering.
An object of the present invention is to provide a kind of base material that can be applied to prepare cell patch.
It is a further object of the present invention to provide a kind of cell sheets are built using Sodium Alginate Hydrogel Films（cell sheet）
Technology.
Still a further object of the present invention is to provide the application of the technology that cell patch is prepared using Sodium Alginate Hydrogel Films.
Above-mentioned purpose of the present invention is achieved through the following technical solutions：
The Sodium Alginate Hydrogel Films that a kind of base material prepared used in cell patch, i.e. surface are modified, by preparing as follows
Method is prepared：Chemical modification is carried out to sodium alginate first, to introduce necessary reactive group, recycle freeze-drying and
Diaphragm is made in sodium alginate by addition drilling agent method, finally utilize large biological molecule to sodium alginate substrate surface or ontology into
Row is cross-linking modified.
Preferably, the chemical modification includes but not limited to the methods of sodium periodate oxidation or EDC/NHS grafting.
Preferably, the perforating agent is sodium chloride solution etc..
Preferably, the large biological molecule is including but not limited to gelatin, collagen or amino acid polypeptide.
More specifically, above-mentioned base material is prepared by following methods：
S1. it weighs sodium alginate to be dissolved in pure water, sodium metaperiodate is added, is protected from light and is stirred to react 1~2 h；
S2. ethylene glycol is added in the solution obtained to S1, the reaction was continued 0.1~2 h；
S3. the solution after S2 being reacted carries out dialysis 3~7 days, frequently changes water；
S4. after dialysing, vacuum freeze drying, the sodium alginate aoxidized；
S5. the sodium alginate and sodium alginate for weighing oxidation are configured to the mixing that total sodium alginate concentration is 1~5%w/v
Sodium chloride is added into mixed solution for solution, makes final concentration of 2 mol/L or less of sodium chloride；
S6. S5 products therefroms are added in the hole of culture plate, after being placed in -80 DEG C of 5~18 h of freezing, vacuum freeze drying；
S7. by S6, treated that culture plate is placed in submerges complete in absolute ethyl alcohol, then is slowly added into 2% calcium chloride solution pair
Material is crosslinked, and finally, makes that the remaining calcium chloride solution in material of removal is washed with deionized；
S8. after S7 treated culture plate sterilizings, it will be carried out using large biological molecule cross-linking modified, obtains to surface and changes
The Sodium Alginate Hydrogel Films of property, as prepare the base material used in cell patch；
Wherein, it is described it is carried out by large biological molecule it is cross-linking modified include following 3 in the way of：
（1）COL-I solution is added to be crosslinked；
（2）EDC/NHS is added and impregnates 12 h, after adding 12 h of gelatin, finally adds EDC/NHS, shaking table mixing into
Row crosslinking；
Or（3）The RGD of pH4.5 ~ 4.7 is added, then a small amount of addition EDC/NHS, shaking table mixing are crosslinked by several times.
Wherein it is preferred to which the amount of the substance of sodium metaperiodate described in step S1 accounts for the amount of the substance of sugar unit in sodium alginate
20~80%.
Preferably, the amount of the substance of sodium metaperiodate described in step S1 accounts for 80% of the amount of the substance of sugar unit in sodium alginate.
Preferably, sodium metaperiodate described in step S1 is the sodium periodate solution of 0.1~2 mg/mL.
Preferably, the speed stirred described in step S1 is 200~500rpm, preferably 300rpm.
Preferably, ethylene glycol described in step S2 is equal with the amount of the substance of the sodium metaperiodate in S1.
Preferably, the quality of the sodium alginate and sodium alginate that are aoxidized described in step S5 is equal.
Preferably, 2% calcium chloride solution described in step S7 is equal with the volume of absolute ethyl alcohol.
Preferably, the dosage of calcium chloride solution described in step S7 ensures calcium ion：Sodium alginate=1：1.（With calcium ion
The gel hardness of the increase of ratio, production becomes larger, but fragile；And with the increase of sodium alginate ratio, the gel of production
Flexibility is good, but hardness is small）.
Preferably, 0.5~1 mLS5 products therefroms are added in every hole of culture plate described in step S6；It is cultivated described in step S8
0.5~1 mL COL-I solution is added in every hole of plate.
Preferably, crosslinking described in step S8 is 4 DEG C of 24~48h of crosslinking.It is highly preferred that using sterile water wash after crosslinking 3 times.
Preferably, sterilizing described in step S8 is to impregnate 3 h, then natural air drying with ethyl alcohol.
Preferably, COL-I solution described in step S8 is the COL-I solution for being dissolved in glacial acetic acid of 0.1~0.3% w/v.More
Preferably, a concentration of 0.2 mmol/L of glacial acetic acid used, COL-I solution concentrations used are 1mg/mL.
The EDC/NHS is 4 mg/ml：The EDC/NHS of 1mg/ml；A concentration of 0.4% w/w of the gelatin；The RGD
A concentration of 0.1 mg/ml.
In addition, the sodium alginate used in the present invention is a kind of alginates, tests and find by numerous studies, most algae
Hydrochlorate（Such as sodium alginate, calcium alginate, potassium alginate, ultra-low viscosity sodium alginate, ultra-low viscosity potassium alginate）It is all suitable for
It is best in the effect of the present invention, wherein sodium alginate.Alginates（Alginate）It is a kind of polysaccharide separated from seaweed
Polymer is by mannite uronic acid（D-mannuronate）And l-gulonic acid（L-guluronate）Composition
Copolymer can act on the hydrogel to form open lattice in the presence of divalent ion such as calcium ion by ionomer.Calcium alginate
Hydrogel plasticity is good, can be made in advance variously-shaped, but its mechanical strength and calcium concentration and concentration of alginate are related, research
Show alginate can be helped to form porous gel structure when calcium ion slowly infiltrates into alginate, and can lead to
The amount for crossing control alginate solution controls the volume to form gel.
Application of the base material of the above-mentioned preparation of the present invention in preparing cell patch also protection scope of the present invention it
It is interior.When applied to cell patch is prepared, this layer of base material can be removed by simple physical and chemical method, finally built
Whole cell patch；Damage of the removal base material to cell and extracellular matrix can be reduced to greatest extent in this process
Wound.
A technique for cell patch being prepared using Sodium Alginate Hydrogel Films, method is as follows：
（1）Sodium Alginate Hydrogel Films after crosslinking prepared by claim 1 are impregnated in cell culture medium, are put into
CO2Incubator is incubated inoculating cell after 1~2 h；
（2）After inoculating cell, co-cultures 5~14 days, 1~2 mL sodium citrate solutions are then added, by sodium alginate water
Gel dissolves, that is, obtains cell patch.
Preferably, step（1）The cell culture medium is the culture medium suitable for cultivating inoculating cell（According to cell type
Difference be adjusted；When being inoculated with people source P3 for osteoblast, the culture medium that uses is containing 10 % FBS without phenol red DMEM/
F12）.
Preferably, step（1）The inoculum density of the cell is 30000~100000/cm2.（The embodiment of the present invention
By taking osteoblast as an example, cell behaviour source P3 is for osteoblast）
Preferably, step（1）The CO2CO in incubator2Volumetric concentration be 5%.
Preferably, step（2）A concentration of 50~70mM of sodium citrate in the sodium citrate solution；More preferably 55
mM。
Application of the cell patch being prepared according to the above method in preparing tissue engineering material is also the present invention's
Within protection domain.
The present invention passes through numerous studies and exploration, first with freeze-drying and adds drilling agent method by sodium alginate system
At diaphragm, in order to introduce necessary reactive group, sodium alginate needs by chemical modification to include but not limited to sodium metaperiodate oxygen
The methods of change and EDC/NHS grafting, using large biological molecule including but not limited to gelatin, collagen, amino acid polypeptide to alginic acid
Sodium substrate surface or ontology are modified, inoculating cell.Finally, after building the cell inoculation of cell patch on material, lead to
The regulation and control for crossing culture environment promote cell Proliferation and generate a large amount of extracellular matrixs, to form complete cell patch, then again
Base material sodium alginate is dissolved using certain density sodium citrate solution, it is final to obtain completely with good raw
The cell patch of object performance.
Cell sheets are prepared using the above-mentioned technology of the present invention, based on CST, the three-dimensional knot of application cell lamella superposition structure
The tissue of the tissue of structure and the method structure three-dimensional structure of cell sheets combination holder, shows in terms of tissue engineered bone structure
Great potential is gone out, the development for inherently promoting organizational engineering is combined with traditional organizational engineering method.
The invention has the advantages that：
The invention discloses one kind can be used for building cell sheets（cell sheet）Base material, this layer of substrate material
Material has soluble characteristic, can be removed by simple physical and chemical method, for building complete cell patch, this process
In can reduce damage of the removal base material to cell and extracellular matrix to greatest extent.
In addition, the present invention is that one kind can be applied to organizational project and repaiies using the method that above-mentioned base material prepares cell sheets
Multiple cell patch technology, the cell patch being prepared are that can save intact cell epimatrix（ECM）Cell sheets, not only
Avoid the digestion in conventional method due to enzyme influences caused by cell, also overcomes the existing behaviour of existing cell patch technology
Make complicated problem.
Cell patch prepared by the present invention has complete ECM, the advantages of combining extracellular matrix, with simple membrane material
Material is compared, and applicability is extensive, is suitable for a large amount of cells, has good biocompatibility, is easy to cell adhesion increment, has good
Good mechanical mechanics property rises in value conducive to the migration of cell, differentiation, and donor is in short supply when being expected to solution organ transplant and transplanting is arranged
The problem of reprimand, can be effectively promoted the reparation of defect, have important meaning to the development of organizational engineering.
Cell sheets are prepared using the technology of the present invention, based on CST, application cell lamella superposition structure three-dimensional structure
Tissue and cell sheets combination holder method structure three-dimensional structure tissue, shown in terms of tissue engineered bone structure
Great potential is combined with traditional organizational engineering method the development for inherently promoting organizational engineering.
Moreover, the method operating procedure that the present invention prepares cell patch is simple, and it is at low cost, it is not necessarily to large scale equipment, is had very
Good application prospect.
Description of the drawings
Fig. 1 is the front and back infrared spectrogram of sodium alginate oxidation.
Fig. 2 is Sodium Alginate Hydrogel Films base material SEM observation charts.
Fig. 3 is sodium alginate holder（Sodium Alginate Hydrogel Films base material）On cell growth status.
Fig. 4 is the variation that sodium citrate is added after cell is co-cultured with base material.
Fig. 5 is that the HE of cell patch is dyed.
Specific implementation mode
It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are purchased in market.
The oxidation of 1 sodium alginate of embodiment
Sodium alginate is polysaccharose substance, has cis- vicinal diamines structure.When being reacted with sodium metaperiodate, sodium metaperiodate can
To promote the C-C keys of sodium alginate to be broken, vicinal diamines structure is made to be oxidized to the higher adjacent aldehyde radical structure of reactivity.New functional group
The sodium alginate surface modification that is introduced as of aldehyde radical provides more means, is a kind of common biomaterial method of modifying.
1,0.2 g sodium alginates are weighed, and are dissolved in the pure water of 10 mL.A certain amount of sodium metaperiodate is added, keeps away
Light is stirred to react 1 h, 300 rpm of mixing speed.After 1h, the second two with amounts of substances such as sodium metaperiodates is added into solution
Alcohol, the reaction was continued 0.5 h.After completion of the reaction, bag filter is added in the above solution to dialyse 3 days, during which frequently changes water.Dialysis finishes
Afterwards, it is freeze-dried using vacuum freeze drier, for use.The ratio between sodium metaperiodate and the amount of substance of sodium alginate sugar unit are respectively
It is 80%, is denoted as OSA80.
2, it takes dry oxidized sodium alginate OSA80 to be put into clean agate mortar, fine powder is milled to bar is ground,
Sample and potassium bromide are pressed 1:100 mass ratio is uniformly mixed, levigate and tabletted, uses Fourier transformation infrared spectrometer
Infrared spectrogram is measured, unoxidized dry sodium alginate does same treatment.
Fig. 1 is that the front and back infrared spectrogram wherein (a) of sodium alginate oxidation be before aoxidizing,（b）After oxidation.1616
cm-1Place is observed that the asymmetric stretching vibration peak of carboxyl, 1415 cm-1For the symmetrical stretching vibration peak of carboxyl.And
1730 cm-1Place is it is observed that the sodium alginate after oxidation has the formation at new aldehyde radical peak here.Show sodium metaperiodate oxygen
Change sodium alginate and produces new aldehyde radical.
The preparation of 2 Sodium Alginate Hydrogel Films base material of embodiment
1, weigh the sodium alginate of certain mass with etc. quality OSA80（Prepared by embodiment 1, the sodium alginate of oxidation）Match
It is set to the mixed solution that total sodium alginate concentration is 2% w/v, meanwhile, sodium chloride is added into mixed solution, keeps sodium chloride dense eventually
Degree is 1 mol/L.After mixing, 6 well culture plates are added in above-mentioned solution, per hole 0.5mL, are placed in -80 DEG C of freeze overnight postpositions
Be freeze-dried in vacuum freeze drier, simultaneously prepared under equal conditions be not added sodium chloride material and be lyophilized as pair
According to.
2, material is fixed on sample stage, metal spraying processing is placed in the vacuum chamber of cold field emission scanning electron microscope, 15 kV
It is observed under voltage, obtains SEM observation charts.As shown in Fig. 2, wherein A, B are the sodium alginate base material for adding pore-foaming agent, C,
D is sodium alginate base material.
Comparison finds that the base material prepared using freeze-drying forms many cavernous structures, is in semi-connected state.Add
The substrate of sodium chloride pore-foaming agent is added many apertures occur compared with the base material for being not added with pore-foaming agent, aperture is in 15 mm or so, greatly
About 60 mm of the aperture in hole.The sodium alginate base material of addition pore-foaming agent is formed by the small hole composite construction of macropore-, in aperture
Many cells were limited on scale and go deep into material internal growth, and there is important meaning to the adherency of cell on surface topology
Justice.Therefore, the formation for being prepared as cell patch of this material provides possibility.
The surface grafting COL-I of 3 sodium alginate holder of embodiment
1, material prepared by embodiment 2 is placed in absolute ethyl alcohol and submerges complete, then to be slowly added into 2% w/v calcium chloride molten
Liquid is crosslinked and is washed to material using 2% calcium chloride solution after calcium chloride solution to be added and absolute ethyl alcohol is isometric,
Finally, make that the remaining calcium chloride solution in material of removal is washed with deionized.
2, after ethyl alcohol impregnates 3 h sterilizings, natural air drying adds per hole and is dissolved in 0.2 mmol/L glacial acetic acid material again
COL-I solution（1 mg/mL）Sterile water wash 3 times, obtains the seaweed of surface modification after 0.5 mL, 4 DEG C of 24 h of refrigerators crosslinking
Sour sodium hydrogel, as prepares the base material used in cell patch.
4 sodium alginate substrate material surface grafted gelatin of embodiment
1, the material prepared according to the method for embodiment 2 is placed in absolute ethyl alcohol and submerges completely, then is slowly added into 2% w/v
Calcium chloride solution hands over material using 2% calcium chloride solution after calcium chloride solution to be added and absolute ethyl alcohol are isometric
Connection and washing finally make that the remaining calcium chloride solution in material of removal is washed with deionized.
2, for material after ethyl alcohol impregnates 3 h sterilizings, 2 ml EDC/NHS (4 mg/ml are added per hole for natural air drying:1
mg/ml）After impregnating 12 h, after 2 ml, 0.4 % w/w gelatin, 12 h is added, 100 μ l EDC/NHS, shaking table mixing are added
Afterwards, 4 DEG C of refrigerators are placed in and are crosslinked sterile water wash 3 times after 24 h, obtain the Sodium Alginate Hydrogel Films of surface grafting gelatin, as
Prepare the base material used in cell patch.
The preparation of 5 RGD- sodium alginates of embodiment
1, the material prepared according to the method for embodiment 2 is placed in absolute ethyl alcohol and submerges completely, then is slowly added into 2% w/v
Calcium chloride solution hands over material using 2% calcium chloride solution after calcium chloride solution to be added and absolute ethyl alcohol are isometric
Connection and washing finally make that the remaining calcium chloride solution in material of removal is washed with deionized.
2, for material after ethyl alcohol impregnates 3 h sterilizings, 2 ml RGD are added per hole for natural air drying（0.1 mg/ml, pH=
4.5~4.7）, a small amount of by several times that EDC/NHS (4 mg/ml are added:1 mg/ml), shaking table mixing is placed in 4 DEG C of refrigerators and is crosslinked 48 h
Afterwards, sterile water wash 3 times obtain the Sodium Alginate Hydrogel Films of surface grafting RGD, as prepare the substrate material used in cell patch
Material.
Cell growth status on 6 sodium alginate holder of embodiment
1, the Sodium Alginate Hydrogel Films after the crosslinking of the preparation of embodiment 3 ~ 5 are immersed in osteoblasts cultivation base（Culture solution
For DMEM F12, without phenol red, 10% fetal calf serum（FBS）, 1% is dual anti-（PS）In, it is put into 5% CO2Incubator is inoculated with after being incubated 2 h
Human marrow mesenchymal stem cell, inoculum density are 50000/cm2A subculture is changed every three days.
2, after inoculating cell, culture medium in orifice plate is siphoned away after co-culturing 14 days, the 5 μ g/ of 1 mL are added in every Porous materials
The FDA solution of mL is incubated 10 min in incubator, is protected from light as possible；Culture dish is taken out, dyeing liquor is sucked, is cleaned three times with PBS,
It is placed in fluorescence microscopy under the microscope.484 nm of excitation wavelength, 520 nm of launch wavelength.
As a result as shown in Fig. 3, the cell density of material surface growth is high, is in fiber fusiform, and cell growth condition is good.
The structure of 7 cell patch of embodiment
1, sodium citrate is a kind of common mild chelating agent.In this experiment, base material is sodium alginate through calcium
Obtained by ionomer, and sodium citrate has the function of chelating calcium ion, and the timbering material may make to degrade after addition.Work as cell
After growing into certain density, the growth substrate material of cell is handled using sodium citrate, and then is built out and a kind of mild made cell
The method of growth substrate is detached from the form of synusia.
2, specific method is：Base material well prepared in advance is impregnated in cell culture medium, CO is put into2Incubator is incubated
Educate inoculating cell after 1~2 h；After inoculating cell, co-cultures 5~14 days, 1~2 mL sodium citrate solutions are then added, it will be extra large
Mosanom hydrogel dissolves, that is, obtains cell patch.
3, shown in such as Fig. 4 (A-D), after cell co-cultures 14 days with base material, it is added a concentration of 55 mM's of 1 mL per hole
After citric acid three sodium solution (10 mM Hepes), base material can be observed at any time and gradually degrade, finally there is one layer close
Bright shape tablet residual, the process are not influenced by sodium citrate chelation.
As Fig. 4 (E-H) observes institute at any time for sodium citrate solution processing of the material through same concentrations of non-inoculating cell
.With the material identical for being inoculated with cell, sodium citrate after the material degradation of non-inoculating cell to still remaining tablet.
In this experiment, EDC/NHS has been used to carry out the material for being crosslinked and being modified without the surfaces COL I between tropocollagen molecule
Substance of the interior nothing insoluble in sodium citrate, therefore, the tablet of the finding of naked eye is the collagen through EDC/NHS crosslinking gained
Layer.
The HE of 8 cell patch of embodiment is dyed
1, by 7 trisodium citrate of example treated flaky residue spreads over adherency glass slide, 2.5% penta 2 is used
Aldehyde solution impregnates, for use.
2, the glutaraldehyde in above-mentioned exhibition piece is removed, is cleaned using PBS solution.Appropriate bush uniformly dyeing is added dropwise to the exhibition piece
Liquid contaminates 3-8 minutes.After pure water rinsing, 1% w/v hydrochloride alcohol solutions are added.After the secondary rinsing of pure water, 0.6% ammonium hydroxide is added and returns
Indigo plant, after optical microphotograph sem observation to be used is coloured to work(, pure water rinses again.Then appropriate eosin stain, 1-3 points of dye is added
Clock is observed again using light microscope, and confirmation is dyed successfully.It after pure water rinsing, is dehydrated using graded ethanol, is added appropriate two
Toluene and after slightly drying, is added resinene and carries out mounting.Use optical microphotograph sem observation coloration result.
3, result is as shown in Fig. 5, and Yihong dyestuff can all dye cytoplasm and extracellular matrix, show in diaphragm
With the presence of a large amount of extracellular matrixs, a large amount of cell is grown on the residual tablet, and is still remained after treatment a large amount of
Extracellular matrix, the structure of cell patch is achieved.
To sum up result of study is shown, the method for the invention for preparing cell patch using Sodium Alginate Hydrogel Films can successfully be made
It is standby to go out to meet the cell patch of desired tissue engineering cell holder condition；Meanwhile the cell sheets not only have suitable for cell increasing
The three-D space structure grown, break up and adhered to, and have good constituent, has nontoxic, without side-effects, has good life
Object compatibility.
Claims (7)
1. a kind of base material prepared used in cell patch, which is characterized in that be prepared by the following method：
S1. it weighs sodium alginate to be dissolved in pure water, sodium metaperiodate is added, is protected from light and is stirred to react 1~2h；
S2. ethylene glycol is added in the solution obtained to S1, the reaction was continued 0~2h；
S3. the solution after S2 being reacted carries out dialysis 3~7 days, frequently changes water；
S4. after dialysing, vacuum freeze drying, the sodium alginate aoxidized；
S5. the sodium alginate and sodium alginate for weighing oxidation are configured to the mixed solution that total sodium alginate concentration is 1~5%w/v,
Sodium chloride is added into mixed solution, makes the final concentration of 2mol/L or less of sodium chloride；
S6. S5 products therefroms are added in the hole of culture plate, after being placed in -80 DEG C of 5~18h of freezing, vacuum freeze drying；
S7. by S6, treated that culture plate is placed in submerges complete in absolute ethyl alcohol, then is slowly added into 2% calcium chloride solution to material
It is crosslinked, finally, makes that the remaining calcium chloride solution in material of removal is washed with deionized；
S8. after S7 treated culture plate sterilizings, it will be carried out using large biological molecule cross-linking modified, obtains surface modification
Sodium Alginate Hydrogel Films as prepare the base material used in cell patch；
Wherein, it is carried out by large biological molecule described in step S8 cross-linking modified including in the way of in the of following 3 kinds：
（1）COL-I solution is added to be crosslinked；
（2）EDC/NHS is added and impregnates 12 h, after adding 12 h of gelatin, finally adds EDC/NHS, shaking table mixing is handed over
Connection；
Or（3）The RGD of pH4.5 ~ 4.7 is added, then a small amount of addition EDC/NHS, shaking table mixing are crosslinked by several times.
2. base material according to claim 1, which is characterized in that the amount of the substance of sodium metaperiodate described in step S1 accounts for seaweed
The 20~80% of the amount of the substance of sugar unit in sour sodium；The amount phase of ethylene glycol described in step S2 and the substance of the sodium metaperiodate in S1
Deng.
3. base material according to claim 1, which is characterized in that the sodium alginate and sodium alginate aoxidized described in step S5
Quality it is equal.
4. base material according to claim 1, which is characterized in that 2% calcium chloride solution and absolute ethyl alcohol described in step S7
Volume is equal.
5. base material according to claim 2, which is characterized in that COL-I solution described in step S8 is 0.1~0.3% w/v
The COL-I solution for being dissolved in glacial acetic acid；Crosslinking described in step S8 is 4 DEG C of 24~48h of crosslinking.
6. base material according to claim 1, which is characterized in that 0.5~1 is added in every hole of culture plate described in step S6
ML S5 products therefroms；0.5~1 mL COL-I solution is added in every hole of culture plate described in step S8.
7. application of any base material of claim 1~6 in preparing cell patch, which is characterized in that prepare cell
The method of diaphragm includes the following steps：
（1）Any base material of claim 1~6 is impregnated in cell culture medium, CO is put into2Incubator is incubated 1~2
Inoculating cell after h；
（2）After inoculating cell, co-cultures 5~14 days, 1~2 mL sodium citrate solutions are then added, by Sodium Alginate Hydrogel Films
Dissolving, that is, obtain cell patch.
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| CN107998453B (en) * | 2017-12-12 | 2020-09-25 | 中山大学附属第一医院 | Surface-modified acellular matrix and modification method thereof |
| CN108956221B (en) * | 2018-04-10 | 2021-08-10 | 上海交通大学附属第一人民医院松江分院 | Method for pathological section preparation by adopting sodium alginate |
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| CN114605664B (en) * | 2020-12-17 | 2023-07-04 | 赣南师范大学 | Hydrogel and preparation method thereof |
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