CN105085938A - Bletilla striata polysaccharide hydrogel, culture medium and application thereof as well as method of inducing differentiation of umbilical cord mesenchymal stem cells to corneal epithelial cells - Google Patents
Bletilla striata polysaccharide hydrogel, culture medium and application thereof as well as method of inducing differentiation of umbilical cord mesenchymal stem cells to corneal epithelial cells Download PDFInfo
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Abstract
The invention relates to the field of cell culture, in particular relates to letilla striata polysaccharide hydrogel, a culture medium and application thereof as well as a method of inducing differentiation of umbilical cord mesenchymal stem cells (hUC-MSCs) to corneal epithelial cells. The method comprises the steps of sulfating letilla striata polysaccharide and then crosslinking with Epsilon-polylysine carrying polyfunctional groups to prepare the polysaccharide hydrogel. The letilla striata polysaccharide hydrogel is of a semi-transparent membrane, has good porosities, and is suitable for the growth and the differentiation of cells. The experiment shows that by taking the letilla striata polysaccharide hydrogel provided by the invention as a scaffold, after the hUC-MSCs and amniotic epithelial cells are subjected to co-culture for 7 days, the differentiation rate of the hUC-MSCs can be up to 31.96%. The differentiation rate (p is less than 0.01) is obviously superior to that of a contrasting example which does not adopt the hydrogel scaffold. In addition, the hydrogel provided by the invention can promote the cells to grow vigorously, and shorten the induction time.
Description
Technical field
The present invention relates to field of cell culture, particularly relate to the method that bletilla striata polyose water gelatin, culture medium and application thereof and inducing umbilical cord mesenchymal stem break up to corneal epithelial cell.
Background technology
Sound corneal epithelial cell is the essential condition that cornea resists extraneous various adverse factor damage, and the limbal stem cell that structure and fuction perfects is the main source of corneal epithelial regeneration.Multiple paathogenic factor, as: ocular injury, operation wound, inflammation, drug toxicity all can cause corneal limbus to damage, and cause limbal stem cell dysfunction, cause epithelial cell to lack, thus cause the danger increasing corneal infection, perforation, neovascularization.Therefore, a large amount of urgently to be resolved hurrily for this difficult problem of epithelial cell of corneal restoration by vitro culture acquisition.Clinical study shows, achieves good efficacy by vitro cultured corneal limbal stem cells as the seed cell of corneal epithelium reparation.But, nutrient solution complicated component required by corneal limbal stem cell autograft in-vitro cultivation, the unstable easily differentiation of cell, cell can not continue amplification, cell phenotype changes, so each transplanting all will take off part corneal limbal tissue on the normal cornea of patient or its family members carry out vitro culture, complex operation, and bring secondary painful to patient, and there is investigator to find when getting confession cornea edge tissue and being greater than 2/3rds, by the irreversible decline of eye surface diseases and eyesight causing strong eye, Most patients is made to be difficult to accept.Therefore, the cell derived finding new autologous and heteroplastic transplantation is necessary very much.
The advantages such as the low stain that umbilical cord mesenchymal stem cells (hUC-MSCs) has, source abundance, molecular marker for increased proliferation, and umbilical cord mesenchymal stem cells does not limit by any ethics and legal principle, has thus attracted numerous investigators.But external evoked hUC-MSCs is very difficult to corneal epithelial cell differentiation.
Dimensional culture technology is by analogue body inner cell growth microenvironment, set up between cell and the extensive connection of extracellular matrix, by forming certain 3-D solid structure, promote that cell is similar to genetic expression in body, extracellular matrix secretion and cell biological function active, therefore it there is very large development prospect stem cell at regenerative medicine and clinical translational medicine application aspect.Complete Three-dimensional cell culture system needs to be made up of three parts, and support, seed cell, culture systems are indispensable.The research of timbering material in recent years shows, hydrogel material becomes the most popular research direction.
Hydrogel is that a class has wetting ability but can not dissolve in water and swelling material can only occur, and is the ubiquitous class physical form of occurring in nature.Water in hydrogel can make to be dissolved in low molecular weight substance wherein from wherein diffusion, there is the characteristic of film, this diffusion property of gel can realize oxygen, the transmission of nutritive substance and the discharge of metabolic waste, for cells survival provides the environment of a similar biological tissue.The hydrogel of the hydrogel that hydrogel can be divided into natural materials to prepare by material source and synthetic materials hydrogel and both compounds.Natural hydrogel material mainly comprises collagen, chitosan, alginate, agarose, hyaluronic acid etc.Its advantage is that cell signal identity is good, can promote sticking, breed and breaking up of cell, have good biocompatibility and biodegradability; Shortcoming is limited source, is difficult to processing, and be difficult to the identity ensureing different batches material, there is immunogenicity, degradation speed, mechanical property are difficult to the problems such as control.Synthetic water gel is the water-soluble swollen body formed through physics or chemically crosslinked by hydrophilic polymkeric substance.PNIPAM, polyvinyl alcohol, polyoxyethylene glycol and derivative thereof study more synthetic materials.The restriction that synthetic hydrogel material is not originated, easy machine-shaping, can adjust physics, chemistry, biomechanics and degradation property as required.But in practice, have also discovered many shortcomings: it is poor that synthetic materials exists cellular affinity, wetting ability and cell adhesion usually, lack the defects such as cell recognition signal site.
Therefore, one should be researched and developed and possess extracellular matrix function, be applicable to the hydrogel of artificial bionic microenvironment of cell development, and use it for the cultivation of cell and differentiation-inducing.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is the method that provides bletilla striata polyose water gelatin, culture medium and application thereof and inducing umbilical cord mesenchymal stem to break up to corneal epithelial cell.To be that BSP is crosslinked with epsilon-polylysine after sulphation modification obtain bletilla striata polyose water gelatin provided by the invention, and this gel can in vitro for cell provides the microenvironment can pressed close in body.Adopt this gel can significantly improve the differentiation rate of cell to corneal epithelial cell differentiation as stent-induced inducing umbilical cord mesenchymal stem.
The preparation method of bletilla striata polyose water gelatin provided by the invention is: after sulfation, obtain bletilla striata polyose water gelatin by BSP with epsilon-polylysine is crosslinked.
The bletilla striata (Bletillastriata) belongs to per nnial herb for the orchid family bletilla striata, is used as medicine with the stem tuber of drying.The good water absorbability that BSP has and wetting ability, can promote the permeation of oxygen, nutritive substance and other water soluble metabolites, can also strengthen the adhesivity of epithelium and stroma cell simultaneously.The present invention by BSP after sulfation with carry the crosslinked obtained polysaccharide hydrogel of multi-functional epsilon-polylysine.Compared with the hydrogel that bletilla striata polyose water gelatin provided by the invention and BSP solution are formed self, there is better hole, and higher viscosity.Further, this preparation method is simple to operate, and gelation time is short.Bletilla striata polyose water gelatin provided by the invention can be provided as the microenvironment that cell provides similar in body in vitro, thus advantageously in the growth and differ entiation of cell.
In an embodiment of the present invention, the preparation method of BSP is: through water extract-alcohol precipitation after being pulverized by the bletilla striata, gained precipitation is resuspended with water, after the mixture extracting of chloroform and butanols, through 30000D ~ 50000D dialysis, and dry obtained BSP.
In certain embodiments, water extract-alcohol precipitation is specially: by bletilla striata powder with after flooding, extracting solution mixes with aqueous ethanolic solution, the obtained precipitation of hold over night.
In certain embodiments, the temperature of lixiviate is 80 DEG C, and the time of lixiviate is 4h.
In certain embodiments, in aqueous ethanolic solution, the volume fraction of ethanol is 95%.
In certain embodiments, the volume ratio of aqueous ethanolic solution and extracting solution is 1:3.
After the mixed solution extracting of chloroform and butanols, the albumen in extract is removed.Centrifugal rear water intaking phase.
In certain embodiments, in the mixture of chloroform and butanols, the volume ratio of chloroform and butanols is 5:1.
In certain embodiments, the volume precipitating water-soluble rear solution is chloroform and butanol mixture 3 times.
In certain embodiments, the dialyzate that dialysis adopts is distilled water.
In certain embodiments, dry is freeze-drying.
The molecular weight that the present invention obtains BSP is 30000D ~ 50000D.
In certain embodiments, dried BSP is also through purifying.
In certain embodiments, purifying is, after the BSP after freeze-drying being dissolved, cross sephadex G-100 post.
In an embodiment of the present invention, Sulfated method is chlorsulfonic acid-pyridine method.
In certain embodiments, Sulfated reagent is chlorsulfonic acid and pyridine.
In certain embodiments, the volume ratio of chlorsulfonic acid and pyridine is 1:6.
In an embodiment of the present invention, the sulfation of BSP is specially: BSP is dissolved in anhydrous DMF, after stirring at room temperature 30min, add sulfur acidizing reagent (volume ratio of chlorsulfonic acid and pyridine is 1:6), be placed in 60 DEG C of stirring in water bath reaction 3h.
In certain embodiments, after sulfation, adjusted to ph is 7.5, is that dialyzate is dialysed 3d after concentrating under reduced pressure with distilled water, obtains sulfated polysaccharides after dialyzate is concentrated through lyophilize.
In an embodiment of the present invention, the molecular weight of epsilon-polylysine solution is 600Da ~ 4300Da, comprising 25 ~ 30 lysine residues.
In an embodiment of the present invention, be cross-linked and be specially, leave standstill after Sulfated BSP solution is mixed with epsilon-polylysine solution.
In certain embodiments, the solvent of epsilon-polylysine solution is PBS damping fluid, and wherein the massfraction of epsilon-polylysine is 3%.
In an embodiment of the present invention, the solvent of Sulfated BSP solution is PBS damping fluid, and wherein the concentration of sulfation BSP is 200 μ g/mL.
In an embodiment of the present invention, crosslinked temperature is 18 DEG C ~ 35 DEG C, and the time is 20s ~ 40s.
In certain embodiments, the crosslinked time is 30s.
Bletilla striata polyose water gelatin prepared by the method for the invention provides.
Bletilla striata polyose water gelatin provided by the invention is translucent membranaceous, wherein has good hole, the Proliferation and differentiation of suitable cell.Experiment shows, with bletilla striata polyose water gelatin provided by the invention for support, by hUC-MSCs and amniotic epithelial cells Dual culture after 7 days, the differentiation rate of hUC-MSCs can reach 31.96%.And Growth of Cells is vigorous, circular in ball.Cell adhesion, on support, is bundled together with support; Be amplified to 2000 times, visible significantly cell, its " pseudopodium " clenches standoff gap.
The bletilla striata polyose water gelatin prepared of the method for the invention provides cell cultures or differentiation-inducing in application.
In an embodiment of the present invention, be induced to differentiate into inducing umbilical cord mesenchymal stem to break up to corneal epithelial cell.
The invention provides the method that inducing umbilical cord mesenchymal stem breaks up to corneal epithelial cell, take bletilla striata polyose water gelatin as support, under nutrient solution existence condition, by umbilical cord mesenchymal stem cells and amniotic epithelial cells Dual culture; Nutrient solution comprises basic medium, Urogastron and Regular Insulin.
In an embodiment of the present invention, the mass ratio of Urogastron and Regular Insulin is (5 ~ 25): (5000 ~ 25000).
As preferably, the mass ratio of Urogastron and Regular Insulin is 2:3000 ~ 3:2000.
Preferably, the mass ratio of Urogastron and Regular Insulin is 1:1000.
In an embodiment of the present invention, the content of Urogastron is 5ng/mL ~ 25 μ g/mL; The content of Regular Insulin is 5ng/mL ~ 25 μ g/mL.
As preferably, the content of Urogastron is 5ng/mL; The content of Regular Insulin is 5 μ g/mL.
As preferably, the content of Urogastron is 10ng/mL; The content of Regular Insulin is 10 μ g/mL.
As preferably, the content of Urogastron is 25ng/mL; The content of Regular Insulin is 25 μ g/mL.
In an embodiment of the present invention, basic culture solution behaviour stem cell serum-free culture medium and epithelial cells serum free medium, the volume ratio of human stem cell serum free medium and epithelial cells serum free medium is (50 ~ 60): (44 ~ 50).
As preferably, the volume ratio of human stem cell serum free medium and epithelial cells serum free medium is 55:45.
The human stem cell serum free medium that the present invention is used and epithelial cells serum free medium can be self-control and also can be purchase acquisition, and the present invention does not limit this, and it is implemented all within protection scope of the present invention.Wherein, human stem cell serum free medium is suitable for culturing stem cells, and the human stem cell serum free medium that the present invention adopts is LonzaUltraCULTURE
tM.Epithelial cells serum free medium is suitable for cultivating epithelial cell, and the epithelial cells serum free medium that the present invention adopts is KSFM nutrient solution.The basic culture solution that the present invention adopts neither containing allogeneic serum, reduces the risk that animal source serum brings to human body.
In an embodiment of the present invention, penicillin and Streptomycin sulphate is also comprised in substratum provided by the invention.
In certain embodiments, the content of penicillin is 100U/mL, and the content of Streptomycin sulphate is 100U/mL.
In an embodiment of the present invention, Dual culture is Indirect co-culture.
In an embodiment of the present invention, umbilical cord mesenchymal stem cells and the number of amniotic epithelial cells are than being (1 ~ 1.5): (4 ~ 6).
Cell Co culturing Techenique is by the technology of two or more cell co-culture in same environment, due to the advantage that it has better antimer environment, is therefore applied in INVENTIONModern cell research.Indirect co-culture system is about in 2 or in 2, above cell is inoculated on different carriers respectively, and then these two kinds of carriers being placed in same culture environment, is that different types of cell shares same culture system and directly do not contact.Indirect co-culture can adopt transwell cell.
In an embodiment of the present invention, umbilical cord mesenchymal stem cells was 3rd ~ 5 generations; Described amniotic epithelial cells is 2nd generation.
In an embodiment of the present invention, the primary separation method of umbilical cord mesenchymal stem cells is tissue mass cell culture; The primary separation method of amniotic epithelial cells is enzyme digestion.
Concrete, the primary separation method of hUC-MSCs comprises the following steps:
Step 1: by the umbilical cord PBS damping fluid rinsing containing 100U/mL penicillin and 100U/mL Streptomycin sulphate 2 times, after the aqueous ethanolic solution being 75% soaks 1min ~ 2min, removes umbilical cord adventitia and blood vessel with volume fraction;
Step 2: cultivate with human stem cell serum-free medium, culture condition is 5%CO
2, 37 DEG C, humidity 95%; Within 5th ~ 7 days, half amount changes substratum, continue cultivation after 12 ~ 14 days full dose change liquid and remove tissue block, collecting cell Secondary Culture.
Concrete, the primary separation method of amniotic epithelial cells comprises the following steps:
Step 1: by amnion with after the PBS damping fluid rinsing containing 100U/mL penicillin and 100U/mL Streptomycin sulphate 2 times, broken is 0.25% tryptic digestion with massfraction;
Step 2: after collagenase II and Dnas enzymic digestion, cultivates.
The concentration of collagenase II is 2.5g/L; The concentration of Dnas enzyme is 0.05g/L; The condition of digestion is 37 DEG C, and 200rpm shakes, and the time is 15min ~ 30min.
The nutrient solution cultivated is the epithelial cells serum free medium comprising Urogastron; The concentration of its Concentrations of Epidermal Growth Factor is 10ng/mL ~ 15ng/mL.
The inoculum density cultivated is 1 × 10
7individual/L.
The condition of cultivating is 5%CO
2, 37 DEG C, after humidity 95% cultivates 2h, after removing not adherent cell, continue cultivation 24 hours.
In an embodiment of the present invention, the time of Dual culture is 7 days.
The invention provides the method that bletilla striata polyose water gelatin, culture medium and application thereof and inducing umbilical cord mesenchymal stem break up to corneal epithelial cell.The present invention by BSP after sulfation with carry the crosslinked obtained polysaccharide hydrogel of multi-functional epsilon-polylysine.This bletilla striata polyose water gelatin is translucent membranaceous, wherein has good hole, the Proliferation and differentiation of suitable cell.Experiment shows, with bletilla striata polyose water gelatin provided by the invention for support, by hUC-MSCs and amniotic epithelial cells Dual culture after 7 days, the differentiation rate of hUC-MSCs can reach 31.96%.This differentiation rate significantly (p<0.01) is better than the comparative example not adopting gel stent.Further, gel provided by the invention can also make Growth of Cells vigorous, shortens induction time.
Accompanying drawing explanation
Fig. 1 shows the hUC-MSCs cellular form (100 ×) through transmitting for 3 ~ 5 generations;
Fig. 2-a shows the amniotic epithelial cells form (40 ×) of original cuiture 12h;
Fig. 2-b shows the amniotic epithelial cells form (40 ×) of Secondary Culture 12h;
Fig. 2-c shows the amniotic epithelial cells HE coloration result (100 ×) of Secondary Culture 24h;
Fig. 2-d shows that the amniotic epithelial cells of Secondary Culture 24h take CK7 as antibody mediated immunity groupization qualification (100 ×);
Fig. 2-e shows that the amniotic epithelial cells of Secondary Culture 24h take CK8 as antibody mediated immunity groupization qualification (100 ×);
Fig. 2-f shows that the amniotic epithelial cells of Secondary Culture 24h take CK18 as antibody mediated immunity groupization qualification (100 ×).
Embodiment
The invention provides the method that bletilla striata polyose water gelatin, culture medium and application thereof and inducing umbilical cord mesenchymal stem break up to corneal epithelial cell., those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can change methods and applications herein or suitably changes and combination not departing from content of the present invention, spirit and scope, realizes and applies the technology of the present invention.
The instrument that the present invention adopts is all common commercially available product, all can buy in market.
The antibody identified for corneal epithelial cell in the embodiment of the present invention is AE1 and AE5, and they are all anti-cytokeratin Ab.AE1 can identify that relative molecular mass is respectively the acidic cell Keratin sulfate of 56500,50000,48000 and 40000, the acid Keratin sulfate that can be 48000 with a kind of relative molecular mass of corneal epithelial cell reacts, with silk-like proteins no cross reactions such as vimentin, desmin, glial fibrillary acidic protein and neurofilament proteins.AE5 can with the Keratin sulfate K3 of corneal epithelial cell (relative molecular mass is 64000) specific binding, respond with the corneal epithelial cell of people, rabbit, ox, and with skin epithelial cell no cross reaction.Cytokeratin K3 is the marker protein of the corneal epithelial cell that differentiation state is higher.Therefore, if when the immunohistochemical staining of cell AE1 and AE5 is positive, so this cell is corneal epithelial cell.
Below in conjunction with embodiment, set forth the present invention further:
The preparation of embodiment 1 BSP
Bletilla striata medicine materical crude slice grinding powder filters after dispersing and dissolving 4h in 80 DEG C of distilled water, elimination impurity.
Filtrate is spent the night via 95% alcohol immersion of 3 times of volumes, and throw out distilled water is resuspended.
Add the chloroform of 1/3 volume and mixed solution (the volume ratio 5:1 of butanols.) be mixed centrifugal extract proteins composition, extracts the liquid of aqueous phase layer.
Through 30000D-50000D dialysis cartridge dialysis (dialyzate is distilled water), make lyophilized powder, obtain BSP crude product.Obtained BSP is further purified through sephadex G-100 by after BSP dissolving crude product.
The sulfation of embodiment 2 BSP
Embodiment 1 is obtained BSP chlorsulfonic acid-pyridine method and carry out sulphation modification.Concrete steps are:
0.2g polysaccharide powder is dissolved in the anhydrous DMF of 35ml, after stirring at room temperature 30min, adds 17.5ml sulfur acidizing reagent (volume ratio of chlorsulfonic acid and pyridine is 1:6), be placed in 60 DEG C of stirring in water bath reaction 3h.Be cooled to room temperature after reaction terminates, reaction solution adds 100ml frozen water, is neutralized to pH7.5 with 1mol/LNaOH, is evaporated to finite concentration, uses distill water dialysis 3d, obtains sulfated polysaccharides after dialyzate is concentrated through lyophilize.
The preparation of embodiment 3 BSP gel
Sulfation BSP obtained for embodiment 2 is dissolved in PBS, is mixed with the polysaccharide soln that concentration is 200 μ g/ml.Be dissolved in by epsilon-polylysine in PBS, massfraction is 3%, obtained linking agent.Polysaccharide soln is mixed with volume ratio 1:1 with linking agent, left at room temperature 30s, translucent membranaceous bletilla striata polyose water gelatin can be formed.
The separation and Culture of embodiment 4 umbilical cord mesenchymal stem cells
Umbilical cord (Serological testing such as hepatitis B, the third liver, HIV, mycoplasma, syphilis is feminine gender) is placed in PBS rinsing containing 100U/mL penicillin and 100U/mL Streptomycin sulphate 2 times, add 75% alcohol of precooling, soak 1-2min, period does not stop to stir umbilical cord; Add PBS rinsing and wash away alcohol 2 times.With tissue shear, umbilical cord is cut into the segment of about 2mm, longitudinally opens with eye scissors for every section, remove three blood vessels (two artery, a vein) in umbilical cord with vascular clamp, remove umbilical cord adventitia simultaneously; The umbilical cord eye scissors of separator well is cut into the tissue block of about 1mm3 size, gets and put into the sterile petri dish that diameter is 10cm in right amount, cover the floorage of 70% culture dish.Add LONZA human stem cell serum-free medium (LonzaUltraCULTURE
tM), 5%CO
2, 37 DEG C, humidity is the CO of 95%
2cultivate in incubator.Within 5th day ~ 7 days, half amount changes substratum, and continuation cultivation 12 days ~ about 14 days full doses are changed liquid and removed tissue block collecting cell Secondary Culture simultaneously.
Through transmitting the hUC-MSCs cellular form in 3 ~ 5 generations as shown in Figure 1, hUC-MSCs growth is vigorous, and border is smooth, presents nido growth, and colony is mostly in polygon or ellipse, and the arrangement of colony inner cell is tight, obscure boundary; Cell boundaries is clear, and endochylema is abundanter, and nucleus is large, and kernel is large.
Get the 3rd generation logarithmic phase cell, cellular segregation liquid digest, Flow cytometry surface antigen CD105, CD45, CD34, CD31, CD40, CD29, CD44, HLA-DR expression, Cell-Quest software analysis result.Each sample analysis 6000 ~ 8000 cells.Result is as shown in table 1:
Table 1 the 3rd generation hUC-MSCs cell surface marker the positive expression rate
| Cell phenotype | CD29 | CD31 | CD34 | CD40 | CD44 | CD45 | CD105 | HLA-DR |
| hUC-MSCs | 94.94±1.06 | 3.27±0.39 | 2.13±0.35 | 1.24±0.06 | 91.8±1.27 | 1.22±0.05 | 99.84±0.17 | 2.93±0.21 |
Through Flow cytometry, as shown in table 1, the 3rd generation hUC-MSCs high expression level CD105, CD44 and CD29, low expression or the marker such as non-expression of HLA-DR, CD31, CD45, CD40 and CD34.
Prepared by embodiment 5 amniotic epithelial cells
People's amnion takes from healthy puerpera (Serological testing such as hepatitis B, the third liver, HIV, mycoplasma, syphilis is feminine gender), amnion is placed in PBS rinsing containing 100U/mL penicillin and 100U/mL Streptomycin sulphate 2 times, is cut into about 30-50cm
2the tissue block of size, add q.s 0.25% tryptic digestive juice, at 37 DEG C, digestion 15 ~ 30min in 200R constant temperature oscillator, every 10min taking-up hand acutely rocks, the centrifugal 5min of 2000rpm, add enough 2.5g/L collagenase II+0.05g/LDnas enzymes again, 4h is digested at 37 DEG C, centrifugal, add containing epithelial cells serum free medium (Keratinocyteserum-freemedium, KFSM) again, the nutrient solution of 10-15ng/ml Urogastron (EGF), piping and druming is even, with 1 × 10
7it is in the culture dish of 10cm that individual/L is inoculated in diameter, putting volume fraction is gently by not adherent cell sucking-off after 5%CO2,37 DEG C of cell culture incubators interior cultivation 2h, because amniotic epithelial cells is adherent slower, the mesenchymal cell mixed or inoblast comparatively fast adherent, at this moment sucking-off again cultured cells (namely difference sticks the cell that method obtains) more than 90% is amniotic epithelial cells.Continue to be cultured to after cytogamy reaches 80% ~ 90%, go down to posterity.The cell Microscopic observation (40 times) as shown in Fig. 2-a of original cuiture 12h.The cell Microscopic observation (40 times) as shown in Fig. 2-b of Secondary Culture 12h.Take out after 2nd generation amniotic epithelial cells being cultivated 24h and (dye Microscopic observation as Fig. 2-c through HE, amplify 100 times), immunohistochemical methods identification of cell Keratin sulfate CK7 (Fig. 2-d), CK8 (Fig. 2-e) and CK18 (Fig. 2-f).
Result shows, Microscopic observation 2nd generation amniotic epithelial cells is rounded, bright, refractivity is comparatively strong, be rich in stereoscopic sensation.The primary cell more than 90% sticking method cultivation through difference is human amnion membrane, the mesenchymal cell mixed or inoblast less, can to sink adherent growth in inoculation 12h, after 2d, cell enters exponential phase of growth, cell quantity showed increased, in irregular polygon, about 4 ~ 5d 90% cytogamy is in blocks, forms individual layer (see Fig. 2-a).The human amnion membrane gone down to posterity can be comparatively fast adherent, gets final product adherent and start growth after inoculation 4 ~ 6h, and after inoculation 24h, Growth of Cells accelerates, and the flat change of cell is large, and nucleus is clear, is positioned at kytoplasm central authorities.About 3 ~ d cell 90% merges, in typical paving stone sample outward appearance (see Fig. 2-c).Hematorylin eosin stains, visible cell size is more homogeneous, and in irregular polygon, kytoplasm enriches, and karyon indigo plant dye, circular, nuclear membrane is clear, and kernel is obvious, visible 2 ~ 3 nuclear fissions (Fig. 2-c).Immunohistochemical methods is identified, see CK7, CK8, CK18 mono-clonal keratin antibody stained positive in the endochylema of the human amnion membrane that the present embodiment is cultivated, endochylema is brown color, and karyon is bluish voilet.Show successfully separation and Culture amniotic epithelial cells.
Embodiment 6
BSP gel is prepared in the lower room of transwell cell.Method is as embodiment 3.0.5mL polysaccharide soln is added six orifice plates, and add isopyknic linking agent, room temperature leaves standstill 30s.
The 3-5 getting propagation logarithmic phase, for hUC-MSCs and 2nd generation amniotic epithelial cells, is inoculated in Transwell co-culture system, and it is 4 × 10 that amniotic epithelial cells inoculum density is implanted in upper room
5individual/hole, hUC-MSCs is implanted in lower room, and inoculum density is 1 × 10
5individual/hole.Every hole adds 2.5ml containing 100U/mL penicillin and 100U/mL Streptomycin sulphate, and volume fraction is 55%LONZA human stem cell serum free medium (LonzaUltraCULTURE
tM), the nutrient solution of 45% epithelial cells serum free medium (Keratinocyteserum-freemedium, KFSM), 10ng/ml Urogastron (EGF), the complete culture solution of 10ug/ml Regular Insulin.
Every the nutrient solution that 2-3 days more renew.Cultivate and take out cell climbing sheet after 7 days.
Observe under inverted microscope: after cultivating 7d, Growth of Cells is vigorous, balling-up round shape, arrangement is tight, visible many colonies; Continue to be cultured to 14d, cell volume obviously increases.
Scanning electron microscopic observation: cultivate visible cell after 7 days and adhere on support, be bundled together with support; Be amplified to 2000 times, visible significantly cell, its " pseudopodium " clenches standoff gap.
Carry out the immunohistochemical staining of cytokeratin AE1 and AE5 and P63 according to the specification sheets of immunohistochemical staining SP method test kit, DAB develops the color.Immunohistochemical staining result shows, visible a large amount of DAB pigmented cells.In be dispersed in distribution and also cell color darker.
Embodiment 7
BSP gel is prepared in the lower room of transwell cell.Method is as embodiment 3.0.5mL polysaccharide soln is added six orifice plates, and add isopyknic linking agent, room temperature leaves standstill 30s.
The 3-5 getting propagation logarithmic phase, for hUC-MSCs and 2nd generation amniotic epithelial cells, is inoculated in Transwell co-culture system, and it is 4 × 10 that amniotic epithelial cells inoculum density is implanted in upper room
5individual/hole, hUC-MSCs is implanted in lower room, and inoculum density is 1 × 10
5individual/hole.Every hole adds 2.5ml containing 100U/mL penicillin and 100U/mL Streptomycin sulphate, and volume fraction is 55%LONZA human stem cell serum free medium (LonzaUltraCULTURE
tM), the nutrient solution of 45% epithelial cells serum free medium (Keratinocyteserum-freemedium, KFSM), 25ng/ml Urogastron (EGF), the complete culture solution of 25ug/ml Regular Insulin.
Every the nutrient solution that 2-3 days more renew.Cultivate and take out cell climbing sheet after 7 days.
Observe under inverted microscope: after cultivating 7d, Growth of Cells is slow, visible a little colony; Be cultured to 14d, cell volume has sees increase.
Scanning electron microscopic observation: continue cultivation after 7 days visible cell adhere on support, be bundled together with support; Be amplified to 2000 times, visible significantly cell, its " pseudopodium " clenches standoff gap.
Carry out the immunohistochemical staining of cytokeratin AE1 and AE5 according to the specification sheets of immunohistochemical staining SP method test kit, DAB develops the color.AE1 immunohistochemical staining result shows, visible a little AE1 antibody DAB pigmented cells, in being dispersed in distribution, and cell color is lighter.AE5 antibody is light painted.
Embodiment 8
BSP gel is prepared in the lower room of transwell cell.Method is as embodiment 3.0.5mL polysaccharide soln is added six orifice plates, and add isopyknic linking agent, room temperature leaves standstill 30s.
The 3-5 getting propagation logarithmic phase, for hUC-MSCs and 2nd generation amniotic epithelial cells, is inoculated in Transwell co-culture system, and it is 4 × 10 that amniotic epithelial cells inoculum density is implanted in upper room
5individual/hole, hUC-MSCs is implanted in lower room, and inoculum density is 1.5 × 10
5individual/hole.Every hole adds 2.5ml containing 100U/mL penicillin and 100U/mL Streptomycin sulphate, and volume fraction is 55%LONZA human stem cell serum free medium (LonzaUltraCULTURE
tM), the nutrient solution of 45% epithelial cells serum free medium (Keratinocyteserum-freemedium, KFSM), 5ng/ml Urogastron (EGF), the complete culture solution of 5ug/ml Regular Insulin.
Every the nutrient solution that 2-3 days more renew.Cultivate and take out cell climbing sheet after 7 days.
Observe under inverted microscope: after cultivating 7d, Growth of Cells is slow, visible a little colony; Be cultured to 14d, cell volume has sees increase.
Scanning electron microscopic observation: continue cultivation 7 days afterwards visible a small amount of cell adhesion on support, be bundled together with support; Be amplified to 2000 times, visible significantly cell, its " pseudopodium " clenches standoff gap.
Carry out the immunohistochemical staining of cytokeratin AE1 and AE5 according to the specification sheets of immunohistochemical staining SP method test kit, DAB develops the color.Immunohistochemical staining result shows, visible a little AE1 antibody DAB pigmented cells.In being dispersed in distribution, and cell color is lighter.AE5 antibody is light painted.
Comparative example 1
The 3-5 getting propagation logarithmic phase, for hUC-MSCs and 2nd generation amniotic epithelial cells, is inoculated in Transwell co-culture system, and it is 4 × 10 that amniotic epithelial cells inoculum density is implanted in upper room
5individual/hole, hUC-MSCs is implanted in lower room, and inoculum density is 1 × 10
5individual/hole.
Culture plate does not add any gel, and every hole adds 2.5ml containing 100U/mL penicillin and 100U/mL Streptomycin sulphate, and volume fraction is 55%LONZA human stem cell serum free medium (LonzaUltraCULTURE
tM), the nutrient solution of 45% epithelial cells serum free medium (Keratinocyteserum-freemedium, KFSM), 10ng/ml Urogastron (EGF), the complete culture solution of 10ug/ml Regular Insulin.
Every the nutrient solution that 2-3 days more renew.Cultivate and take out cell climbing sheet after 7 days.
Observe under inverted microscope: after cultivating 7d, cell is then in fusiformis, and single layer of confluent grows.Continue to be cultured to 14d, visible cell volume has sees increase.
Carry out the immunohistochemical staining of cytokeratin AE1 and AE5 and P63 according to the specification sheets of immunohistochemical staining SP method test kit, DAB develops the color.Immunohistochemical staining result shows, visible a little DAB pigmented cells.In be dispersed in distribution and also cell color more shallow.
Embodiment 9
To embodiment 6 ~ 8 and comparative example 1 gained cell after immunohistochemical methods detects, count the cell of AE1, AE5 dyeing, the differentiation rate calculating two groups compares.Often organize sampling 3 times, statistic data after detecting respectively.
Differentiation-inducing rate=staining positive cells number/total cellular score × 100%.
Statistics is as table 2:
Table 2 is group cytodifferentiation rate respectively
Note: the different letter representation of same column shoulder mark has significant difference (p<0.01)
Experiment shows, by umbilical cord mesenchymal stem cells and amniotic epithelial cells Dual culture, after 7 days, umbilical cord mesenchymal stem cells part is differentiation due, and cell is in being dispersed in distribution, and after testing, the differentiation rate that hUC-MSCs is divided into corneal epithelial cell can reach 31.96%.And do not adopt the differentiation rate of hUC-MSCs in the comparative example of gel stent to be 26.52%; Visible, method provided by the invention can improve the percentage that hUC-MSCs breaks up to corneal epithelial cell, and this effect has significant difference (p<0.01).
Below be only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. the preparation method of bletilla striata polyose water gelatin, is characterized in that, obtains bletilla striata polyose water gelatin by BSP after sulfation so that epsilon-polylysine is crosslinked.
2. preparation method according to claim 1, is characterized in that, the preparation method of described BSP is: through water extract-alcohol precipitation after being pulverized by the bletilla striata, gained precipitation is resuspended with water, after the mixture extracting of chloroform and butanols, through 30000D ~ 50000D dialysis, dry obtained BSP.
3. preparation method according to claim 1, is characterized in that, described Sulfated reagent is the mixture of chlorsulfonic acid and pyridine.
4. preparation method according to claim 1, is characterized in that, described crosslinked temperature is 18 DEG C ~ 35 DEG C, and the time is 20s ~ 40s.
5. the bletilla striata polyose water gelatin that described in any one of Claims 1 to 4, preparation method obtains.
6. the bletilla striata polyose water gelatin that described in any one of Claims 1 to 4, preparation method obtains cell cultures or differentiation-inducing in application.
7. application according to claim 6, is characterized in that, described in be induced to differentiate into inducing umbilical cord mesenchymal stem and break up to corneal epithelial cell.
8. the inducing umbilical cord mesenchymal stem method of breaking up to corneal epithelial cell, it is characterized in that, the bletilla striata polyose water gelatin obtained with preparation method described in any one of Claims 1 to 4 for support, under nutrient solution existence condition, by umbilical cord mesenchymal stem cells and amniotic epithelial cells Dual culture; Described nutrient solution comprises basic medium, Urogastron and Regular Insulin.
9. method according to claim 7, is characterized in that, the mass ratio of described Urogastron and Regular Insulin is (5 ~ 25): (5000 ~ 25000).
10. method according to claim 7, is characterized in that, described umbilical cord mesenchymal stem cells is (1 ~ 1.5) with the number ratio of amniotic epithelial cells: (4 ~ 6).
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