CN103642750A - Dynamic enriching method of epidermic cells - Google Patents
Dynamic enriching method of epidermic cells Download PDFInfo
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- CN103642750A CN103642750A CN201310613989.9A CN201310613989A CN103642750A CN 103642750 A CN103642750 A CN 103642750A CN 201310613989 A CN201310613989 A CN 201310613989A CN 103642750 A CN103642750 A CN 103642750A
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Abstract
The invention relates to a dynamic enriching method of epidermic cells, and belongs to the technical field of biological cells. The method comprises the following steps: carrying out pretreatment, digesting and liquid preparation, taking epidermis to prepare a plurality of epidermic pieces, placing the epidermic pieces in a digestion container, and adding a digestive enzyme for dynamic digestion to obtain a cell suspension; mixing the cell suspension obtained by digestion with protein gels, uniformly mixing to prepare a liquid and spraying the liquid on a wounded surface. The dynamic enriching method provided by the invention is applied to wound repair and has the advantages of high culture efficiency, convenience in operation, high skin source utilization ratio and the like.
Description
Technical field
The dynamic enriching method that the present invention relates to a kind of epidermic cell, belongs to biomass cells technical field.
Background technology
The sickness rate of China burn is total population 2～10 ‰, and nearly ten million people of burns victims occurs every year, need reaching of hospital care hundreds thousand of more than, and serious burn person's mortality ratio is very high.Burn is often left over a large amount of scars after curing, and to patient's appearance, the bodily form and function, psychological aspects, all can cause negative impact.Wherein, large-area burns cause serious skin disappearance, are important factors that causes death, the key of dealing with problems of Pi Yuan.
In order to overcome the problem of Pi Yuan, normally adopt the method for closing as early as possible burn wound, closing in early days not only of burn wound can be saved patient's life, also be the important means that alleviates scar, in existing technology, mainly by microparticle skin technology, cultivation epidermic cell and Recell technology, realize.
(1) microparticle skin technology needs first from patient's body not burned part to take off healthy skin source, use physical method to be dispersed into the little particulate of trying one's best in this limited autologous skin source again, then Hui Zhi is to patient's the surface of a wound, this technology can expand skin graft 10～20 times, but the method adopts physical method that skin graft is pulverized, and then Hui Zhi is to the surface of a wound, and the skin grain attaching rate of transplanting is not high, therefore, effect is difficult to guarantee;
(2) time that the method for cultivation epidermic cell need be waited for is longer, and late result is not good;
(3) Recell technology is by pancreatin, stratum basale epidermic cell to be digested under static state, then utilize spray method that the liquid epidermic cell being suspended in ringer's solution is sprayed to the surface of a wound, use this technology skin graft can be expanded to 40～60 times, but defect is, the method need to consume a large amount of high concentration degradation enzymes, in treating processes, can adopt scraping to wait physics mode sampling, and the physical methods such as scraping can be different because of operator's skills involved in the labour, cell has certain infringement, the cell viability obtaining by Recell is only in 70% left and right, therefore, the actual amount to obtain of epidermal stem cells is difficult to confirm, simultaneously, because cell is to be in a liquid state to be sprayed at damaged skin surface, cell occurs to flow, be difficult to guarantee cell being uniformly distributed at wound face, the healing of burn wound also cannot guarantee thus, need to repeatedly spray and could finally realize the recovery of burn wound, extended treatment flow process, and whole spraying external member used still be take import equipment at present as main, the price of separate unit is between 1-2 ten thousand, treatment cost also improves accordingly greatly.
How in view of this, the inventor furthers investigate making full use of the problem in limited skin source, develops a kind of dynamic enriching method of epidermic cell, and this case produces thus.
Summary of the invention
In order to overcome the above-mentioned problems in the prior art, the invention provides that a kind of culture efficiency is high, the dynamic enriching method of simple operation, epidermic cell that skin source utilization ratio is high.
For achieving the above object, the technical scheme that the present invention takes is as follows:
The dynamic enriching method of epidermic cell, comprises pre-treatment, digestion and dosing, and concrete steps are,
(1) pre-treatment: get epidermis, make some epidermis sheets;
(2) digestion: get step (1) gained epidermis sheet and be placed in digestion vessel, add digestive ferment, dynamically digest, obtain cell suspension;
(3) dosing: will digest gained cell suspension and mix with protein gelatin, and mix dosing.
For realizing better result of use, further arranging of technique scheme is as follows:
In step (1), the epidermis of getting is split-thickness skin graft, and the particle diameter of every of making epidermis sheet is at 1-5mm.
In step (2), every 1-2cm
2split-thickness skin graft adds the digestive ferment of 4-6ml, and in described digestive ferment, Main Function composition is pancreatin and EDTA, and wherein, the mass concentration of pancreatin in digestive ferment is 0.25-0.5%, and the mass concentration of EDTA in digestive ferment is 0.01-0.05%; Dynamically digestion is carried out under constant temperature, the standing and dynamic two states hocketing, consist of, first standing 5-7 minute (also can be described as static digestion), then enters dynamically, dynamically keep after 3-7 minute, after standing 5-7 minute, enter dynamically, temperature is controlled at 35-38 ℃ again, be preferably 36.8-37.5 ℃, standing-dynamically-standing-one of dynamic construction circulation, each dynamic digestive process comprises 2-5 this circulation, carries out cell and wash away after dynamically having digested.Preferably, the intermediate split thickness skin graft that the thickness of split-thickness skin graft is 0.3-0.6mm; The rotating speed of guaranteeing material in digestion vessel in the time of is dynamically 60-300rpm, and preferably rotating speed is 180-300rpm, and the reagent that cell washes away employing is any in PBS, standard physiological saline or Ge Linshi liquid, till going out digestion vessel completely with cell.
Between step (3) and step (2), at least once filter, adopting order number is to filter under 20-60 condition, removes epidermis residue, and the enchylema of lower floor is for dosing.Preferably, in lower floor's enchylema, add inhibitor and form cell suspension, inhibitor is preferably autoserum or soybean trypsin, and in every 5-15ml lower floor enchylema, autoserous interpolation 1-3 drips (every 15 is 1ml), soybean trypsin addition be lower floor's cell liquid measure 0.8-3 doubly.
In step (3), protein gelatin is preferably Fibrin Glue, and mixing process is: respectively cell suspension is mixed with setting accelerator, Fibrin Glue.Preferably, setting accelerator and Fibrin Glue first carry out pre-mixing, again cell suspension and setting accelerator/Fibrin Glue are poured in the different cylinders of dual barrel syringe, under same pressure-acting, cell suspension and setting accelerator/Fibrin Glue are extruded simultaneously, mix dosing, in every 5-10ml Fibrin Glue, added 1-3 to drip (every 15 is 1ml) setting accelerator, the total amount of Fibrin Glue and setting accelerator and cell suspension equivalent.
In technique scheme, the steps such as described pre-treatment, digestion and dosing dynamically digest instrument or biological culture with carrying out on shaking table at epidermis, epidermic cell dynamically digests instrument and comprises base, and be arranged on control panel, digestion vessel, filtration vessel, mixing vessel, control unit, well heater, vibrator, sensor and the power supply on base, wherein, control panel comprises display screen and button, and vibrator is arranged on the bottom of digestion vessel, makes digestion vessel produce the vibration of certain frequency; Well heater is arranged on the outside surface of digestion vessel, for the liquid in hot digestion container, temperature sensor is arranged on digestion vessel, for monitoring the Heating temperature of digestion vessel, above-mentioned well heater, vibrator are connected with the output terminal of control unit respectively, control panel is connected with the input terminus of control unit respectively with sensor device, and power supply is respectively control panel, control unit, well heater and vibrator power supply.
Due in epidermic cell except epidermal stem cells, other cells have the function of propagation equally, contribute to wound healing, therefore, in such scheme of the present invention, whole split-thickness skin grafts are originated as epidermic cell, whole dynamic digestive process is under 35-38 ℃ of constant temperature, adopt biological shaking table (in experimentation, can select conventional biology laboratory cultivation shaking table, if model is HS-100C), vibrator (cultivate medication with biology laboratory and create identical by vibrator effect, structure is different, thereby rotating speed also there are differences, conventionally need conversion) etc. mode dynamic condition is provided, can obtain on the one hand larger, more cell concentration, simultaneously, the working concentration of digestive ferment and usage quantity also can reduce greatly, digestive process can complete in tens minutes, compare with conventional digested overnight mode, shortened digestion time, more be conducive to the maintenance of cytoactive, reduce cell depletion, adopting the dynamic digestion techniques acquisition epidermic cell in the present invention is a kind of reliable method, the epidermic cell having broken up of dosing gained is sprayed on the surface of a wound, epidermic cell at the surface of a wound by the contrary epidermal stem cells that is differentiated to form, promote wound healing, Pi Yuan is used adequately reasonably, amplification times reaches 100-120 doubly.
Wherein, the definition of split-thickness skin graft refers to the parts of skin more than subcutis of skin, comprises razor graft (thickness is in 0.3mm left and right) and intermediate split thickness skin graft (thickness is about 0.3-0.6mm).
Accompanying drawing explanation
Fig. 1 is the plan structure schematic diagram of epidermic cell enriching instrument used in the embodiment of the present invention 2;
Fig. 2 is the perspective view of epidermic cell enriching instrument used in the embodiment of the present invention 2;
Fig. 3 is for adopting gained cell of the present invention to be applied to after the surface of a wound of nude mice back the coloration result contrast (under 100 power microscopes) of the 2nd week;
Fig. 4 is for adopting gained cell of the present invention to be applied to after the surface of a wound of nude mice back the coloration result contrast (under 100 power microscopes) of the 3rd week;
Fig. 5 is for adopting gained cell of the present invention to be applied to after the surface of a wound of nude mice back the coloration result contrast (under 100 power microscopes) of the 4th week;
Fig. 6 is that the epidermic cell of the work that under microscope, static digestion obtains obtains enrichment (under 100 times of phase microscopes);
Fig. 7 is that the inoblast that under microscope, dynamically digestion obtains obtains enrichment (under 100 times of phase microscopes).
Number in the figure: base 1; Base upper surface 11; Base lower surface 12; Operator's console 13; Control panel 2; Display screen 21; Button 22; Digestion vessel 3; Filtration vessel 4; Thrust-augmenting nozzle 41; Mixing vessel 5; Control unit 6; Well heater 7; Vibrator 8; Sensor 9; Power supply 10.
Embodiment
In the present embodiment, the dynamic enriching method of epidermic cell, comprises pre-treatment, digestion and dosing, and concrete steps are,
(1) pre-treatment: get 2cm
2split-thickness skin graft, wherein, tomography cortex be tomography thickness be 0.3mm in thick epidermis, split-thickness skin graft is made to the epidermis sheet that diameter is 2-3mm size;
(2) digestion: in the time of pre-treatment split-thickness skin graft, by control panel, start epidermic cell enriching instrument, by epidermic cell enriching instrument preheating 5-10 minute, holding temperature is at 36.8-37.5 ℃, step (1) gained epidermis sheet is placed in to digestion vessel, the digestive ferment that adds 5ml, wherein, the main component that plays katalysis in digestive ferment is pancreatin and EDTA, in digestive ferment, the content of pancreatin is 0.25%(mass percent), the content of EDTA is 0.01%(mass percent), after standing 7 minutes, (realization of its rotating speed can be cultivated with shaking table and be realized by biology laboratory by control panel, to adjust the rotating speed of the epidermis sheet in digestion vessel, also can adopt Fig. 6-), dynamically start, dynamichandling after 3 minutes standing 7 minutes again, standing-dynamically-standing-one of dynamic formation circulation, under constant temperature, circulate after 3 times, adopt PBS to wash away cell, washing away duration is 10-30s, cell is washed away after digestion vessel completely, termination is washed away, it is that in 40 object filtration vessels, (in the present embodiment, the filter element of filtration vessel is selected nylon screen that the solution obtaining is transferred to order number, also can select the filter screen of equal order number or play the filtration unit of same purpose) filter, remove after epidermis residue, in filtering gained lower floor enchylema, add 1-2 and drip autoserum or 15ml STI, obtain cell suspension,
(3) dosing: will digest gained cell suspension and mix with Fibrin Glue, that is: first setting accelerator and Fibrin Glue are carried out to pre-mixing, the addition of setting accelerator is that 3-4 drips, again cell suspension and setting accelerator/Fibrin Glue are poured in the different cylinders of dual barrel syringe, under same pressure-acting, cell suspension and setting accelerator/Fibrin Glue are extruded simultaneously, mixed dosing, dosing gained differentiation epidermic cell is coated on burn wound, and forms the thick membranaceous thin layer of 0.5-1mm on surface of a wound surface.
The present embodiment is identical with setting and the principle of work of embodiment 1, difference is: the steps such as the pre-treatment of the present embodiment and digestion dynamically digest on instrument at specific equipment epidermic cell carries out, in conjunction with Fig. 6 and Fig. 7, comprise base 1, and be arranged on the control panel 2 on base 1, digestion vessel 3, filtration vessel 4, mixing vessel 5, control unit 6, well heater 7, vibrator 8, sensor 9 and power supply 10 etc., wherein, control panel 2 comprises display screen 21 and button 22, in the present embodiment, above-mentioned display screen 21 adopts charactron to show, be mainly used in displays temperature, vibrational frequency, the parameters such as cycling time, button 22 comprises power initiation button and vibration unlatching button, vibrator 8 is arranged on the bottom of digestion vessel 3, the vibration that makes digestion vessel 3 produce certain frequency, in the present embodiment, described vibrator 8 adopts vibrating motor, the speed range of vibrating motor is 1100-2500rpm, cultivate and use the function of shaking table identical with biology laboratory in embodiment 1, be the solution making in digestion vessel 3 and produce the vibrations of certain frequency or rock, well heater 7 is arranged on the outside surface of digestion vessel 3, for hot digestion container 3, the solution in digestion vessel 3 is remained between 36.8-37.5 ℃, sensor 9 is arranged on the through hole of digestion vessel 3 sidewalls, the probe of temperature sensor stretches in solution, the Heating temperature of Real-Time Monitoring digestion vessel 3, temperature sensor 9 can adopt the temperature sensor of Multiple Type, and object is accurately to sense the temperature of digestion vessel 3 interior solution, well heater 7, vibrator 8 are connected with the output terminal of control unit 6 respectively, control panel 2 is connected with the input terminus of control unit 6 respectively with sensor device 9, power supply 10 is respectively control panel 2, control unit 6, well heater 7 and vibrator 8 power supplies, in the present embodiment, above-mentioned control unit 6 adopts microprocessor, can select the microprocessor of a plurality of models, as long as can realize above-mentioned control function, the upper end of filtration vessel 4 is provided with filtering net, the inlet portion of digestion vessel 3, filtration vessel 5 and mixing vessel 6 and control panel 2 are arranged on respectively on the upper surface 11 of base 1, on base 1 upper surface 11, are further provided with an operator's console 13, before epidermis digestion, and the pre-treatment such as shearing, control unit 6, power supply 10 and vibrator 8 are arranged on respectively on the lower surface 12 of base 1, and the bottom connection of the top of vibrator 8 and digestion vessel 3 touches.
Adopt the present embodiment said apparatus to carry out the dynamic enrichment of epidermic cell, operation steps is as follows:
(1) pre-treatment: get epidermis, make some particle diameters at 1-5mm epidermis sheet on the operator's console 13 of epidermic cell enriching instrument;
(2) digestion: start epidermic cell enriching instrument power supply 10, get step (1) gained epidermis sheet and be placed in digestion vessel 3, add digestive ferment, by control unit 6, control vibrator 8 and well heater 7, dynamically digest, after dynamically digestion finishes, cell is gone out to digestion vessel, obtain cell solution;
(3) filter: step (2) gained cell solution is proceeded to filtration vessel 4 and filter, remove epidermis residue, lower floor's enchylema is for dosing;
(4) dosing: filtration gained lower floor enchylema is proceeded to mixing vessel, mix with protein gelatin, mixed dosing, in the present embodiment, in lower floor's enchylema, add inhibitor and form cell suspension, inhibitor is autoserum, protein gelatin is Fibrin Glue, concrete mixing process is: setting accelerator and Fibrin Glue first carry out pre-mixing, again cell suspension and setting accelerator/Fibrin Glue are poured in the different cylinders of dual barrel syringe, under same pressure-acting, cell suspension and setting accelerator/Fibrin Glue are extruded simultaneously, have mixed dosing.
The digestive ferment wherein relating to and digestive ferment addition, setting accelerator, inhibitor, the isoparametric setting of protein gelatin can reference example 1 in corresponding parameter, the effect of the present embodiment vibrator is cultivated identical with shaking table effect with biology laboratory in embodiment 1, all to make the solution in digestion vessel 3 produce the vibrations of certain frequency or rock, but apparatus structure is cultivated with shaking table different with principle from biology laboratory in embodiment 1, therefore the speed range of vibrating motor is 1100-2500rpm in the present embodiment, in concrete operations, different according to the selected kind of epidermis, and the difference for the treatment of capacity, the rotating speed of vibrating motor may be selected to be 1100, 1500, 1800, 2300 with different gears such as 2500 grade, it is 30 that filtration vessel can be selected order number, 40, 50 or 60, to adapt to the demand for the treatment of effect and processing intent.
The implementation result of above-described embodiment is carried out to holistic approach.
Wherein, table 1 is total epidermic cell amount to obtain during different digestion method in embodiment 1, and table 2 is the comparison of wound healing time between above-described embodiment gained skin factor of different cell submission group.
Total epidermic cell amount to obtain (P<0.01) of the different digestion methods of table 1
| Digestion method | Epidermis area (cm 2） | There is vigor epidermic cell amount to obtain (10 6） |
| Standing/ |
5 | 2.42±0.16 |
| |
5 | 2.35±0.39 |
| |
5 | 3.26±1.21 |
| |
5 | 15.04±4.82 * |
| |
5 | 24.07±7.25 * |
As can be seen from Table 1, when choosing the tomography epidermis of equal area, under the dynamic condition of rotating speed (rotating speed in table 1 refers to the rotating speed of shaking table for biology laboratory cultivation, model HS-100C) when 180-240rpm, can obtain 15 * 10
6-24 * 10
6cell amount to obtain, the amount to obtain of its vigor epidermic cell is ideal.
Table 2 is the comparison of wound healing time between skin factor of different cell submission group
(
*represent and the comparison of Fibrin Glue group P<0.05;
*represent and Fibrin Glue comparison, P<0.01)
| Group | Number of days ± standard error |
| Fibrin Glue | 15.60±2.98 |
| Microcarrier | 8.00±1.23 * |
| Suspension | 6.80±0.86 ** |
| Static | 7.60±1.08 * |
Adopt the gelatinous enrichment of cell of above-described embodiment gained to be sprayed on burn wound, cell is evenly distributed at the surface of a wound, and the abundance of cell is not less than 4000/cm
2, due in epidermic cell except epidermal stem cells, other cells have the function of propagation equally, contribute to closing of the surface of a wound, therefore, in such scheme, whole split-thickness skin grafts is originated as epidermic cell, in carrying out dynamic digestive process, under optimal temperature, adopt shaking table that dynamic condition is provided, can be conducive on the one hand the active larger cell of screening, thereby obtain larger, more cell concentration, simultaneously, the working concentration of digestive ferment and usage quantity also can reduce greatly, both reduced reagent cost, the reduction of digestive ferment concentration is also conducive to the maintenance of cytoactive, and present method gained archeocyte also can share with other material is fused.Adopting the dynamic digestion techniques acquisition epidermic cell in the present invention is a kind of reliable method, the epidermic cell having broken up can be at the surface of a wound by contrary differentiation, form epidermal stem cells, promote wound healing, Pi Yuan is used adequately reasonably, amplification times reaches 120 times, wherein according to the difference for the treatment of capacity, pancreatin in the digestive ferment of selecting, the concentration of EDTA etc. also increases gradually, to adapt to processing power, the addition of inhibitor is corresponding with pending epidermis amount, while increasing as the amount when epidermis sheet, pancreatin content in digestive ferment is 0.3%, EDTA content is 0.035%, in corresponding digestive process, time of repose is controlled at 6 minutes, dynamic time is controlled at 4 minutes, circulate 4 times, or the pancreatin content in digestive ferment is 0.5%, EDTA content is 0.05%, in corresponding dynamic digestive process, time of repose is controlled at 5 minutes, dynamic time is controlled at 7 minutes, circulate 5 times, dynamically the rotating speed of digestion should be controlled in suitable scope, and too high or height all can affect the amount to obtain of final active cells, thereby in the present invention, rotating speed should be controlled at 180-300rpm, as shown in table 1, and the acquisition amount of its active cells is ideal, when the epidermis sheet adopting is razor graft, its treatment time all will reduce with respect to the intermediate split thickness skin graft treatment time and digestive ferment concentration etc., filtering order number is that 20 orders are with intensive filtration effect, pancreas enzyme concentration in digestive ferment is 0.28%, EDTA concentration is 0.02%, in corresponding digestive process, time of repose is controlled at 5 minutes, dynamic time is controlled at 4 minutes, circulate 3 times, or the pancreas enzyme concentration in digestive ferment is 0.38%, EDTA concentration is 0.04%, and in corresponding dynamic digestive process, time of repose is controlled at 4 minutes, dynamic time is controlled at 6 minutes, circulates 3 times.
The test result of take below while being applied to burn the nude mice back surface of a wound is example, carries out the concrete elaboration of technical solution of the present invention.
Fig. 3, Fig. 4 and Fig. 5 are for adopting after the present embodiment technical scheme, the coloration result contrast of the surface of a wound, wherein Fig. 3 is second week HE coloration result, A is that microcarrier is cultivated epidermic cell, visible epidermis forms, and thicker, but owe evenly, and multiple stratification is obvious, B is uncultivated epidermal cell suspension, the thicker epidermal area of same formation, has significantly stratification again, and C is that former culture epidermic cell has also formed epidermal area, there is significantly stratification again, D is pure fibrin glue, and without obviously epidermal area formation, the surface of a wound has a large amount of inoblasts; Fig. 4 is the 3rd week experimentation on animals histology, HE coloration result: A is that microcarrier is cultivated epidermic cell, there is thicker but inhomogeneous epidermal area to form, B is uncultivated epidermal cell suspension, has thicker epidermal area to form, and more even, C is former culture epidermic cell, have thinner uniform epidermal area to form, D is pure fibrin glue, without obviously epidermal area formation; Fig. 5 is 4th week experimentation on animals histology, HE coloration result: A is that microcarrier is cultivated epidermic cell, there is thicker but inhomogeneous epidermal area to form, B is uncultivated epidermal cell suspension, has thicker epidermal area to form, and more even, C is former culture epidermic cell, epidermal area is thinner, and D is pure fibrin glue, without epidermal area, forms.
From can obviously finding out among Fig. 6 and Fig. 7: the cell (Fig. 6) that the cell obtaining by dynamic digestion (Fig. 7) obviously obtains more than static state digestion, after Trypan Blue, dynamically the cell of digestion still maintains higher cell viability (Fig. 7).
In epidermic cell, except epidermal stem cells, other cells have the function of propagation equally, contribute to wound healing, therefore, in such scheme provided by the present invention, whole split-thickness skin grafts are originated as epidermic cell, in carrying out dynamic digestive process, adopt at a certain temperature mode of vibration that dynamic digestion condition is provided, can obtain on the one hand larger, more cell concentration, simultaneously, the working concentration of digestive ferment and usage quantity also can reduce greatly, can shorten digestion time simultaneously, be conducive to the maintenance of cytoactive, reduce cell depletion, adopting the dynamic digestion method acquisition epidermic cell in the present invention is a kind of reliable method, the epidermic cell having broken up can be at the surface of a wound by contrary differentiation, form epidermal stem cells, promote wound healing, Pi Yuan is used adequately reasonably, amplification times reaches 100-120 doubly.
Above content is the further description of provided technical scheme being done in conjunction with the preferred embodiment of the present invention; can not assert that the concrete enforcement of the present invention is confined to above-mentioned these explanations; for general technical staff of the technical field of the invention; without departing from the inventive concept of the premise; can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (10)
1. the dynamic enriching method of epidermic cell, is characterized in that, comprises pre-treatment, digestion and dosing, and concrete steps are,
(1) pre-treatment: get epidermis, make some epidermis sheets;
(2) digestion: get step (1) gained epidermis sheet and be placed in digestion vessel, add digestive ferment, dynamically digest, obtain cell suspension;
(3) dosing: will digest gained cell suspension and mix with protein gelatin, and mix.
2. the dynamic enriching method of epidermic cell as claimed in claim 1, is characterized in that: in step (1), the epidermis of getting is split-thickness skin graft, and the particle diameter of every epidermis sheet is at 1-5mm.
3. the enrichment of epidermic cell as claimed in claim 2 and application, is characterized in that: the intermediate split thickness skin graft that the thickness of described split-thickness skin graft is 0.3-0.6mm.
4. the dynamic enriching method of epidermic cell as claimed in claim 1, is characterized in that: in step (2), and every 1-2 cm
2split-thickness skin graft adds the digestive ferment of 4-6ml, and described digestive ferment comprises pancreatin and EDTA, and wherein, the mass concentration of pancreatin in digestive ferment is 0.25-0.5%, and the mass concentration of EDTA in digestive ferment is 0.01-0.05%; Dynamically digestion is carried out under constant temperature, the standing and dynamic two states hocketing, consists of, and carries out cell and wash away after dynamically having digested, and obtains cell suspension.
5. the dynamic enriching method of epidermic cell as claimed in claim 4, it is characterized in that: every pair of standing, the circulation of dynamic construction hocketing, while dynamically digesting, first standing 5-7 minute, enter again dynamically, dynamically keep after 3-7 minute, each dynamically digests and comprises 2-5 circulation, and homo(io)thermism is controlled at 35-38 ℃.
6. the dynamic enriching method of the epidermic cell as described in claim 4 or 5, is characterized in that: described guarantee that the rotating speed of material in digestion vessel is 60-300rpm when dynamic; The reagent that cell washes away employing is any in PBS, standard physiological saline or Ge Linshi liquid, till going out digestion vessel completely with cell.
7. the dynamic enriching method of epidermic cell as claimed in claim 6, is characterized in that: the described rotating speed 180-300rpm that guarantees material in digestion vessel when dynamic.
8. the dynamic enriching method of epidermic cell as claimed in claim 1, it is characterized in that: between step (3) and step (2), at least also need once to filter, the order number of filtration is 20-60, removes epidermis residue, and in lower floor's enchylema, add inhibitor, form cell suspension, wherein, inhibitor is autoserum or soybean trypsin, in every 5-15ml lower floor enchylema, autoserous interpolation 1-3 drips, the addition of soybean trypsin be lower floor's enchylema 0.8-3 doubly.
9. the dynamic enriching method of epidermic cell as claimed in claim 1, it is characterized in that, in step (3), protein gelatin is Fibrin Glue, the mixing process of cell suspension and protein gelatin is: respectively by cell suspension and setting accelerator, Fibrin Glue mixes, described setting accelerator and Fibrin Glue first carry out pre-mixing, in every 5-10ml Fibrin Glue, adding setting accelerator 1-3 drips, again cell suspension and setting accelerator/Fibrin Glue are poured in the different cylinders of dual barrel syringe, under same pressure-acting, cell suspension and setting accelerator/Fibrin Glue are extruded simultaneously, the total amount of Fibrin Glue and setting accelerator and cell suspension equivalent, mixed dosing.
10. the dynamic enriching method of epidermic cell as claimed in claim 1, it is characterized in that: described pre-treatment, digestion and dosing dynamically digest instrument or biological culture with carrying out on shaking table at epidermis, epidermic cell dynamically digests instrument and comprises base, and be arranged on control panel, digestion vessel, filtration vessel, mixing vessel, control unit, well heater, vibrator, sensor and the power supply on base, wherein, control panel comprises display screen and button, vibrator is arranged on the bottom of digestion vessel, makes digestion vessel produce the vibration of certain frequency; Well heater is arranged on the outside surface of digestion vessel, for the liquid in hot digestion container, temperature sensor is arranged on digestion vessel, for monitoring the Heating temperature of digestion vessel, above-mentioned well heater, vibrator are connected with the output terminal of control unit respectively, control panel is connected with the input terminus of control unit respectively with sensor device, and power supply is respectively control panel, control unit, well heater and vibrator power supply.
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| PCT/CN2014/076037 WO2015078137A1 (en) | 2013-11-27 | 2014-04-23 | Epidermal cell collecting instrument and dynamic collecting method thereof |
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| CN111849871A (en) * | 2020-08-11 | 2020-10-30 | 广州市麦施缔医疗科技有限公司 | Method for obtaining epidermal cell suspension |
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| WO2015078137A1 (en) * | 2013-11-27 | 2015-06-04 | 鲁建国 | Epidermal cell collecting instrument and dynamic collecting method thereof |
| CN111849871A (en) * | 2020-08-11 | 2020-10-30 | 广州市麦施缔医疗科技有限公司 | Method for obtaining epidermal cell suspension |
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Inventor after: Han Chunmao Inventor after: Hu Xinlei Inventor after: Lu Jianguo Inventor after: Han Yang Inventor before: Han Chunmao Inventor before: Hu Xinlei Inventor before: Lu Jianguo Inventor before: Han Yang |
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