CN108126246A - Artificial skin construction method based on compound stem cell - Google Patents

Abstract

The invention discloses a kind of artificial skin construction methods based on compound stem cell, it is using de- cell amnion as skin tissue engineering scaffold, it plants on the basal surface of amnion, being placed in culture solution liquid culture to build to obtain artificial skin using epidermal stem cells and mesenchymal stem cell as seed cell.The artificial skin that the present invention is built have the advantages that antigenicity is low, skin strength is high, good mechanical property, it is harmless to the surface of a wound and body, can induce the growth of body own cells, wound healing and effect, transplanted for clinic skin and provide donor.

Description

Artificial skin construction method based on compound stem cell

Technical field

The invention belongs to bioengineered tissue technical fields, are related to a kind of construction method of Dermis equivalent, particularly relate to A kind of and method for using amnion as stent, building Dermis equivalent.

Background technology

Mesenchymal stem cell is a kind of relatively inmature cell, has the potential for being divided into epidermis, dermal tissue, It can wound healing.After the mesenchymal stem cell culture to 5 generations of in vitro culture, still with differentiation potential, this is just To be laid a good foundation using the Marrow Mesenchymal Stem Cells artificial skin of culture proliferation.

Epidermal stem cells are the stem cells for having self duplication and multi-lineage potential, its normal proliferative and differentiation is dimension Hold skin and its appendicle (Han Xian ﹑ hairs, sebaceous glands) basic demand of structure and function integrality.Epidermal stem cells can not only Long Term Passages culture in vitro, and its Proliferation, Differentiation potential is kept, and also show to do with embryo under the conditions of certain environment The similar differentiation potential of cell.Therefore, human and animal's epidermal stem cells of purifying are obtained, can be that structure has physiological function Artificial skin provide seed cell.

With being constantly progressive for mankind's technology, the sick and wounded, particularly extensive deep burn patient have no longer met current Clinically commonly use the therapeutic effect that can be provided for the treatment of means, it is desirable to the surface of a wound after reparation on mode of appearance and function will with just Normal skin is close, also it is desirable to which the skin after reparation will have various cutaneous appendages such as hair, sebaceous glands, sweat gland etc. or even wish Can be as excellent as before after skin repair, without any scar.Therefore, artificial skin of the structure with real biological function is realized " perfect skin healing ", it has also become the challenge subjects of organization engineering skin research.

Invention content

The object of the present invention is to provide a kind of artificial skin construction methods based on compound stem cell, a kind of similar to provide In the Dermis equivalent of human skin.

Artificial skin construction method of the present invention is using de- cell amnion as skin tissue engineering scaffold, by epidermis Stem cell and mesenchymal stem cell are planted as seed cell on the basal surface of amnion, are placed in liquid culture in culture solution Artificial skin is obtained with structure.

Specifically, the present invention is that the skin tissue engineering scaffold is soaked in the IMDM culture mediums containing 10% fetal calf serum In, after mesenchymal stem cell and epidermal stem cells are mixed in 1: 1 ratio, it is seeded in stent base face, culture structure Artificial skin containing mesenchymal stem cell and epidermal stem cells.

Wherein, the density for being seeded in the mixing stem cell on stent base face is preferably 2 × 10⁶A/cm²。

The present invention is used for the mesenchymal stem cell of inoculation and epidermal stem cells are the stem cell passed on after 3 generations.

Further, the present invention be the mesenchymal stem cell and epidermal stem cells are added in culture medium be made it is mixed Suspension uses.Closer, of the present invention for the culture medium of preparing suspension is type i collagen and 100mg by 100mg Chondroitin sulfate be dissolved in 100ml IMDM culture mediums, the culture obtained after 0.22 μm of membrane filtration degerming after mixing Base.

The present invention can come from the amnion of various mammals as amnion used in skin tissue engineering scaffold, Preferably, the present invention uses the amnion for coming from people.

And then the present invention handles to obtain as the amnion of skin tissue engineering scaffold using following methods：

Fetal membrane is cleaned up with containing 1% dual anti-PBS liquid, after separating chorion and vascular tissue, then it is dual anti-to contain 1% PBS liquid is rinsed well, is soaked in so that in the diluted 1% Triton X-100 of PBS liquid, 37 DEG C of water bath with thermostatic control oscillation treatments are for 24 hours； PBS rinsed cleans, then be placed in 0.25% trypsase containing 0.02% EDTA, 37 DEG C of water bath with thermostatic control oscillation treatment 4h, PBS drifts Wash 30min；In -80 DEG C of precooling 1h, vacuum freeze drying is for 24 hours；After freeze-drying, with Co⁶⁰Radiation exposure 20min.

Wherein, Co⁶⁰The exposure intensity of ray is 20kGy.

The present invention is using de- cell amnion as skin tissue engineering scaffold, by certain density bone marrow mesenchymal stem cells and table Stent base face is inoculated in after the mixing of skin stem cell, structure obtains artificial skin after a period of time is cultivated in culture solution.Wherein, Using amnion as stent, be on the one hand conducive to the growth of mesenchymal stem cell and epidermal stem cells, on the one hand in skin The tear of artificial skin can be effectively prevent during transplanting, increases the operability of operation.Meanwhile amnion and life after skin graft operation Object tissue has good compatibility, some nutritional ingredients can be drawn from organism in transplant early, to promote skin Healing.

Type i collagen has larger affinity, weaker antigenic, good for amnion surface protein molecule Biocompatibility and biodegradable safety, and degradable absorption, adhesion strength are good.Chondroitin sulfate has anti-inflammatory, acceleration wound Mouth healing, the effect of protection collagenous fibres, can promote fiber in matrix to increase, and enhance permeability, and improvement blood circulation accelerates new Old metabolism promotes the absorption of penetrating fluid and the elimination of inflammation；Its polyanion has strong water-retaining property.The two is combined into use In suspension stem cell, contribute to cell seeding in amnionic basement face, and contribute at dermatoplasty initial stage moisture holding and Reparation to wound.

The organization engineering skin based on epidermal stem cells and mesenchymal stem cell that the present invention is built lacks for skin Damage transplanting, it is insufficient to solve the problems, such as clinically to transplant manually skin-derived, avoids immunological rejection, and at the beginning of dermatoplasty Phase promotes the healing of skin.

The artificial skin that the present invention is built have antigenicity is low, skin strength is high, good mechanical property, to the surface of a wound and body without Evil can induce the advantages of growth of body own cells, wound healing and effect, and donor is provided for clinic skin transplanting.

Description of the drawings

Fig. 1 is that amnion takes off HE dyeing observation results before and after cell.

Fig. 2 is the flow cytometer detection result of mesenchymal stem cell.

Fig. 3 is epidermal stem cells immunofluorescence microscopy result.

Fig. 4 is the scanning electron microscopic observation result taken off with amnion after the compound stem cell of cytoskeleton.

Specific embodiment

Following embodiments are only the preferred technical solution of the present invention, are not used to carry out any restrictions to the present invention.For For those skilled in the art, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made Any modification, equivalent substitution, improvement and etc., should all be included in the protection scope of the present invention.

Embodiment 1.

The fetal membrane that the present embodiment uses is the Cesarean esction fetal membrane obtained in strict accordance with donor Medicine standard, and fetal membrane hepatitis Malicious antibody, syphilis antibody and human immunodeficiency virus's antibody test are feminine gender.

Fetal membrane with the PBS liquid containing dual anti-(penicillin, streptomysin) is rinsed three times repeatedly, surface sludged blood is removed, puts In 4 DEG C containing dual anti-PBS buffer solution in preserve.

Fetal membrane, the chorion of blunt separation removal remaining and vascular tissue are taken out, to be rinsed repeatedly containing dual anti-PBS liquid, It is soaked in behind label matrix face in the diluted 1% Triton X-100 of PBS liquid, in desk-top water-bath constant temperature oscillator, 37 DEG C, Oscillation treatment is for 24 hours repeatedly under the conditions of 100rpm/min.

It after the completion of oscillation, is rinsed, is placed in 0.25% trypsase containing 0.02% EDTA with PBS, 37 DEG C of oscillation treatment 4h After take out, PBS rinsing 30min obtain de- cell amnion.

The fresh amnion piece of 0.5cm × 1cm sizes and de- cell membrane film are taken respectively, are fixed through 10% neutral formalin 12h, successively dehydration, waxdip, embedding, slice and Hematoxylin-eosin dyeing, the de- cell feelings of optical microphotograph Microscopic observation amnion Condition.Before A takes off cell for amnion in Fig. 1, after B takes off cell for amnion.As can be seen from the figure amnion cell after de- cell processing It goes to the greatest extent.

The de- cell amnion smooth expansion in plate that will be rinsed well, is placed in -80 DEG C of refrigerator precooling 1h, vacuum refrigeration Drying for 24 hours, after freeze-drying, is cut into the rectangular fritter of 2cm × 2cm, with the Co of intensity 20kGy⁶⁰X ray irradiation x 20min makes after disinfection Cytoskeleton (HAAM) is taken off into amnion.

Embodiment 2.

The gross area about 60cm prepared by Example 1²Through Co⁶⁰The HAAM that x ray irradiation x is crossed, is placed in 25cm²In culture bottle, IMDM culture medium 10mL are added in, in CO₂It is stood for 24 hours for 37 DEG C in incubator, leaching liquor is prepared, for measuring the cell of HAAM Toxicity.

Third generation mesenchymal stem cell is taken, by 1 × 10³The quantity of a cells/well is inoculated in 96 orifice plates, is inoculated with altogether 4 plates add in the IMDM culture mediums containing 10% FBS, in CO₂It is cultivated for 24 hours in incubator.Culture medium is sucked, is distinguished on every block of plate As a control group, leaching liquor 200 μ L of the addition containing 10% FBS is as experiment by IMDM culture medium 200 μ L of the addition containing 10% FBS Group, every group of 6 holes set 8 repetitions.Setting one is not added with the blank well of stem cell simultaneously in every group.

Since second day, one piece of orifice plate is taken out every 48h, adds in 10 μ L of CCK8 solution, continues to cultivate 1h, takes out cooling To room temperature, 450nm wavelength is selected, each hole absorbance value (OD) is measured in microplate reader.

Each group surveys absorbance value and subtracts practical absorbance of the absorbance value of the corresponding blank well of respectively group as each group Value is counted, and the results are shown in Table 1.As a result withIt represents.

The opposite proliferation rate of cell is calculated according to measured each group absorbance value：Cell is with respect to proliferation rate (RGR)=reality Test a group absorbance value/control group absorbance value.

According to data in table 1, measure the opposite proliferation rate acquired per hole absorbance value through CCK8 methods and show that amnion is through Co⁶⁰ Relative toxicity after x ray irradiation x is very low, in safe range.

Embodiment 3.

Healthy cleaning grade SD rats 2 are taken, are weighed, 0.4% chloraldurate of intraperitoneal injection (0.5mL/100g) is anaesthetized, and is lain on the back Position is fixed on aseptic operation table, and cotton balls dips the double hind legs of iodophor disinfection and abdomen, sterilizes 3 times repeatedly.Across pass under aseptic condition Section removes breastbone, and bilateral lower limb femur, shin bone remove the soft tissue of attachment, and skeleton specimen is rinsed 3 with D-Hank ' s liquid Time.Femur, shin bone both ends are cut, exposure ossis is drawn with 1mL syringes and rinses marrow containing 1% dual anti-IMDM culture mediums For several times, until marrow becomes white from red, single cell suspension is made in piping and druming to chamber.Breastbone part is shredded with scissors, is placed in plane It in ware, is blown and beaten with IMDM culture mediums, draws supernatant liquid in centrifuge tube.

Above-mentioned two groups of liquid 1000r/min are centrifuged into 5min, supernatant are abandoned, to contain 10% FBS, 1% dual anti-IMDM culture mediums It is resuspended, obtains mesenchymal stem cell cell.

By obtained mesenchymal stem cell cell inoculation in culture bottle, 37 DEG C, 5%CO are put₂It is cultivated in incubator. Culture carries out half amount and changes liquid after 3 days, full dose changes liquid after 5 days, in 37 DEG C, 5%CO₂Continue to cultivate in incubator.

Mesenchymal stem cell degrees of fusion is passed on when reaching 90% or so.Culture medium is abandoned, PBS is cleaned 2 times, is added in 0.25% pancreatin (containing 0.02% EDTA) solution 1mL, 37 DEG C of digestion 3min, micro- Microscopic observation cell rounding floating add in culture Base terminates digestion.Cell suspension is moved into centrifuge tube with suction pipe, 1000rpm centrifugations 5min.Supernatant is abandoned, adds in culture medium piping and druming Cell is resuspended, and is passed in 1: 2 ratio, 37 DEG C, 5%CO₂It is cultivated in incubator.

When later mesenchymal stem cell is fused to 90%, method is passed on according to this, until reaching for the 3rd generation, is obtained pure The mesenchymal stem cell of change.

The 3rd generation mesenchymal stem cell of degrees of fusion 90% or so is taken, abandons culture medium, PBS is cleaned 2 times, adds in 0.25% Trypsin solution 1mL, 37 DEG C of digestion 5min, micro- Microscopic observation cell rounding floating, addition culture medium, which terminates, to be digested.It will with suction pipe Cell suspension is moved into centrifuge tube, and 1000rpm centrifugation 5min abandon supernatant, and PBS is cleaned 2 times, centrifuged again.Supernatant is abandoned, is added in Cell suspension, cell count is made in 1mL PBS, piping and druming, and adjustment cell density is 10⁵A/mL.Filtration cell, 1000rpm from Heart 5min abandons supernatant, and it is 200 μ L to add in PBS adjustment liquor capacities, gently bounces cell as suspension, is separately added into CD105- PE, CD90-PE, CD45-FITC antibody, room temperature, which is protected from light, is incubated 30min；Add in 4~5mL PBS cleaning solutions, 1000rpm centrifugations 5min abandons supernatant, adds in 200 μ L buffer solutions, gently bounces cell again as suspension, uses BD FACS Aria III streamings Cell instrument is detected.After being combined with antibody by detecting different cells the difference with fluorescence intensity, analysis cell institute band table Face marker and cell type.

Testing result is as shown in Fig. 2, the 3rd generation mesenchymal stem cell surface marker is shown, mescenchymal stem cell table The expression rate of face molecule CD105, CD90 respectively (99.54 ± 0.13) % and (95.74 ± 1.39) %, hematopoietic cell surface markers The result of object CD45 is negative, expression rate (0.87 ± 1.39) %, it was demonstrated that cultivates cell as mesenchymal stem cell.

Embodiment 4.

Aseptic collection newborn SD rat skin of back is fully cleaned with the dual anti-no calcium and magnesium PBS containing high concentration, with ophthalmic tweezers Gently divest the adipose tissue of skin corium.The epidermal tissue of acquisition is cut into the fritter of 0.1cm × 0.1cm, substrate is face-down It is affixed on 35mm²In culture dish；When epidermis and ware bottom attach it is close and it is not dry when, a small amount of serum-free keratinocyte culture is added dropwise Base (KSFM) infiltrates tissue block, is placed in 5%CO₂, cultivate in 37 DEG C of incubators.After for 24 hours, addition culture solution to normal usage, every other day Change liquid.

When tissue block peripheral cell length is to 1~2cm of diameter, had digestive transfer culture is carried out.Culture solution in culture dish is discarded, with nothing Calcium and magnesium PBS is rinsed 3 times, and 400 μ L, 0.25% trypsase (containing 0.02% EDTA), room temperature digestion about 1min are added in into culture dish Afterwards, pancreatin is discarded；400 μ L, 0.25% trypsase (containing 0.02% EDTA) is added in into culture dish again, after digesting about 5min, It is neutralized with isometric culture solution, piping and druming is uniform.Cell suspension is moved into centrifuge tube, 1000r/min centrifugations 5min.It abandons Clearly, it is blown and beaten uniformly with 1mL culture solutions, is inoculated in culture dish, is put into 37 DEG C, 5%CO₂Quiescent culture 20min in incubator, gently It is light to draw non-attached cell and discard, and culture solution is added into former ware, it is put into 37 DEG C, 5%CO₂It cultivates in incubator, changes every other day Liquid.The 2nd passage is carried out when cell fusion degree reaches 90% or so, abandons culture medium, PBS is cleaned 2 times, adds in 0.25% pancreatin (containing 0.02% EDTA) solution 1mL, 37 DEG C of digestion 3min, micro- Microscopic observation cell rounding floating add in culture medium termination and disappear Change.Cell suspension is moved into centrifuge tube with suction pipe, 1000rpm centrifugations 5min.Supernatant is abandoned, culture medium piping and druming cell is added in and carries out It is resuspended, is passed in 1: 2 ratio, 37 DEG C, 5%CO₂It is cultivated in incubator.It is passed on to obtain the purifying of the 3rd generation in this approach Epidermal stem cells.

The epidermal stem cells for being passaged to for the 3rd generation are taken, suck culture medium, are rinsed 2 times with no calcium and magnesium PBS, are added into culture dish Enter 0.25% trypsase of 1mL (containing 0.02% EDTA), after cell is rounded suspension mostly, add in isometric culture medium With, move in centrifuge tube, 1000rpm centrifugation 5min.Cell is resuspended with 1mL culture mediums, carries out cell count under the microscope, with Per hole 10³A cell seeding is in 96 orifice plates, per hole supplementing culture medium to 200 μ L.Next day sucks the culture medium in orifice plate, uses PBS washs 3min, sucks PBS.Add in 4% paraformaldehyde, 4 DEG C of fixed 30min；Paraformaldehyde is sucked, washs 3 times with PBS, often Secondary 3min.With 0.5% Triton X-100 in the transparent 15min of room temperature, Triton X-100 are sucked, wash 3 times with PBS, every time 3min.30min is closed in 37 DEG C with 10% lowlenthal serum, primary antibody (6 integrin of K15, α) is added in, by 1: 1000 dilution proportion Afterwards, 200 μ L are added in per hole, 4 DEG C are protected from light overnight incubation.

Primary antibody is recycled in centrifuge tube in case next time uses, addition PBS is washed 3 times, each 3min；Fluorescence secondary antibody is pressed 1: After 500 dilution proportions, 200 μ L are added in per hole, 37 DEG C are protected from light incubation 1h；Secondary antibody is sucked, is washed 3 times with PBS, each 3min；It adds in DAPI contaminates core, and room temperature is protected from light 10min；It is washed 2 times with PBS, each 3min；It is thin in fluorescence microscopy Microscopic observation with 10% glycerine mounting Cellular surface fluorescence intensity.

As shown in figure 3, the cell after passage can be by 6 integrin specificity mark of hair follicle stem cell labeling molecule K15 and α Note.Using above method culture cell culture to 3 generation when still have clonality.It is demonstrated experimentally that the present embodiment Method can realize the in vitro culture to epidermal stem cells.

Embodiment 5.

It will be through Co⁶⁰The freeze-drying amnion that x ray irradiation x is crossed is soaked in sterile saline and rinses 5 times；Amnion stroma is faced On be laid in 6 well culture plates, merging IMDM culture mediums infiltration is prewetted 2h；The IMDM containing 10% fetal calf serum is placed in again to train completely It supports base and infiltrates the 2h that prewets.

100mg type i collagens and 100mg chondroitin sulfates are dissolved in 100mL IMDM culture mediums, are configured to mix molten Liquid, with 0.22 μm of filter filtration sterilization.

The 3rd generation mesenchymal stem cell and epidermal stem cells are taken, after abandoning culture medium, are washed twice with PBS respectively, addition contains 0.25% pancreatin of 0.02% EDTA when cell is largely rounded and floats, adds in IMDM complete mediums and terminates digestion, transfer Into centrifuge tube, 1000rpm centrifugations 5min.Supernatant is abandoned, mesenchymal stem cell and epidermal stem cells is made to be mixed with 1: 1 ratio It closes, adds in the culture medium mixed solution 1mL containing type i collagen and chondroitin sulfate, microscopic counting.Using containing type i collagen and sulphur The culture medium of aching and limp ossein adjusts cell concentration to 2 × 10⁶A/mL is configured to 7mL cell suspensions.

Cell suspension is slowly dropped in 6 orifice plates for being covered with amnion, adds 1ml cell suspensions per hole, be statically placed in 37 DEG C, 5%CO₂1~2h is cultivated in incubator, is made on cell adhesion to amniotic material.The IMDM containing 10% fetal calf serum is added in cultivate completely Base 1mL is statically placed in incubator for 24 hours, and stem cell is made fully to be adhered to stent net wall.Supernatant is sucked, culture medium is replaced, in 37 ℃、5%CO₂Quiescent culture in incubator changes a not good liquor in every 3 days.Observe cell growth status, cell life under the microscope daily Length is vigorous.

After 8 days, amnion is taken out, prepared by artificial skin completes.

Micro- Microscopic observation cell growth status, and clip has planted 0.5cm × 1cm size membrane films of cell, plantation The one side of cell is placed upward, and 2.5% 4 DEG C of glutaraldehyde is overnight；PBS is rinsed 3~5 times, 10~15min every time；1% osmic acid is fixed 30min is rinsed 3~5 times, 10~15min every time with PBS；Ethyl alcohol 30%, 50%, 70%, 80%, 90% is each primary, every time 10~ 15min；100% ethyl alcohol is primary, 100% acetone is secondary, every time 20~30min；With acetic acid ester interchange liquid and acetone（1∶1）Mixing Liquid displacement is primary, and the ester interchange liquid displacement of pure acetic acid is primary, each 10~20min, critical point drying about 2~3h.Sample after drying Surface vacuum metal spraying is carried out, the growing state of scanning electron microscopic observation amnion superficial cell selects suitable amplification factor shooting to shine Piece.

As shown in figure 4, plantation mesenchymal stem cell and epidermal stem cells, to amnion surface, cell adherence is on amnion And growth conditions are good, after cultivating 5 days, cell is paved with amnion, radial irregular form.

Claims (10)

1. a kind of artificial skin construction method based on compound stem cell is using de- cell amnion as skin tissue engineering branch Epidermal stem cells and mesenchymal stem cell are planted as seed cell on the basal surface of amnion, are placed in culture solution by frame Middle liquid culture is to build to obtain artificial skin.

2. artificial skin construction method according to claim 1, it is characterized in that the skin tissue engineering scaffold is impregnated In the IMDM culture mediums containing 10% fetal calf serum, mesenchymal stem cell and epidermal stem cells in 1: 1 ratio are mixed and connect Kind is in stent base face, artificial skin of the culture structure containing mesenchymal stem cell and epidermal stem cells.

3. artificial skin construction method according to claim 2, it is characterized in that the mixing being seeded on stent base face is done The density of cell is 2 × 10⁶A/cm²。

4. artificial skin construction method according to claim 1, it is characterized in that the medulla mesenchyma for inoculation is done Cell and epidermal stem cells are the stem cell passed on after 3 generations.

5. artificial skin construction method according to claim 1, it is characterized in that by the mesenchymal stem cell and table Skin stem cell, which is added in culture medium, is made suspension use.

6. artificial skin construction method according to claim 5, it is characterized in that the culture medium for being used to prepare suspension It is to be dissolved in 100ml IMDM culture mediums by the type i collagen of 100mg and the chondroitin sulfate of 100mg, through 0.22 μm after mixing The culture medium obtained after membrane filtration degerming.

7. artificial skin construction method according to claim 1, it is characterized in that the amnion comes from mammal Amnion.

8. artificial skin construction method according to claim 1, it is characterized in that the amnion comes from the amnion of people.

9. artificial skin construction method according to claim 1, it is characterized in that the amnion is frozen through vacuum freeze drying After dry, with Co⁶⁰Radiation exposure 20min.

10. artificial skin construction method according to claim 9, it is characterized in that the Co⁶⁰The exposure intensity of ray is 20kGy。