WO2015078137A1 - 一种表皮细胞富集仪及其动态富集方法 - Google Patents

一种表皮细胞富集仪及其动态富集方法 Download PDF

Info

Publication number
WO2015078137A1
WO2015078137A1 PCT/CN2014/076037 CN2014076037W WO2015078137A1 WO 2015078137 A1 WO2015078137 A1 WO 2015078137A1 CN 2014076037 W CN2014076037 W CN 2014076037W WO 2015078137 A1 WO2015078137 A1 WO 2015078137A1
Authority
WO
WIPO (PCT)
Prior art keywords
digestion
container
epidermal
cell
vibrator
Prior art date
Application number
PCT/CN2014/076037
Other languages
English (en)
French (fr)
Inventor
鲁建国
韩春茂
韩杨
胡信雷
Original Assignee
鲁建国
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201310610927.2A external-priority patent/CN103614295B/zh
Priority claimed from CN201310611230.7A external-priority patent/CN103623001B/zh
Priority claimed from CN201310613989.9A external-priority patent/CN103642750A/zh
Priority claimed from CN201320763903.6U external-priority patent/CN203625377U/zh
Application filed by 鲁建国 filed Critical 鲁建国
Publication of WO2015078137A1 publication Critical patent/WO2015078137A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/48—Automatic or computerized control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/42—Integrated assemblies, e.g. cassettes or cartridges
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/16—Vibrating; Shaking; Tilting
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa

Definitions

  • the invention relates to a cell enrichment instrument and an enrichment method thereof, in particular to an epidermal cell enrichment instrument and a dynamic enrichment method thereof.
  • micro-skin technology the limited auto-derived skin source is physically dispersed into as small particles as possible, and then implanted into the patient's wound surface, this technology can expand the skin 10 to 20 Times, but using this physical method, it is difficult to further enlarge the skin sheet on the one hand, and on the other hand, the adhesion rate of the epidermis is low, and it is difficult to ensure the therapeutic effect;
  • RECELL technology because the closure of the wound is completed by the proliferation of epidermal stem cells, RECELL
  • the technique is to statically digest the epidermal cells of the basal layer, and then spray the epidermal cells on the wound surface by spraying.
  • the technique can theoretically enlarge the skin slices by 40 to 80.
  • epidermal stem cells are mainly present in the basal layer. Therefore, this technique mainly uses epidermal stem cells to repair wounds.
  • Static digestion mainly has the following defects: poor mixing between digestive enzymes and substrates, long digestion time, and uneven digestion.
  • the present inventors conducted research on this, and specially developed an epidermal cell enrichment meter and a dynamic enrichment method thereof, and the present invention was produced.
  • the object of the present invention is to provide an epidermal cell enrichment meter and a dynamic enrichment method thereof, which adopts dynamic digestion to obtain high-density and viable epidermal cells, and the whole process has a short digestion time and uniform digestion.
  • An epidermal cell enrichment apparatus comprising a base, a control panel disposed on the base, a digestion container, a filter container, a mixing container, a control unit, a heater, a vibrator, a sensor, a power source, and the like, wherein the control panel includes a display screen And the button, the vibrator is installed at the bottom of the digestion container, so that the digestion container generates a certain frequency of vibration; the heater is installed on the outer surface of the digestion container for heating the liquid in the digestion container, and the temperature sensor is installed on the digestion container, For monitoring the heating temperature of the digestion vessel, the heater and the vibrator are respectively connected to the output end of the control unit, and the control panel and the sensor are respectively connected to the input end of the control unit, and the power source is a control panel, a control unit, a heater and a vibration respectively. Power supply.
  • a through hole is formed in the side wall of the digesting container for mounting a temperature sensor.
  • the upper end of the filter container is provided with a sieve for filtering the dynamically digested solution, and the mesh number of the filter is 20-60.
  • the inlet portion of the digestion vessel, the filtration vessel, and the mixing vessel, and the control panel are respectively mounted on the upper surface of the base.
  • an operation table is further disposed on the upper surface of the base for pretreatment such as shearing before skin digestion.
  • the vibrator uses a vibration motor.
  • the control unit, the power source and the vibrator are respectively mounted on the lower surface of the base, and the top of the vibrator is in contact with the bottom of the digestion container.
  • a method for dynamically enriching epidermal cells including pretreatment, digestion, filtration and dosing, the specific steps are: (1) Pretreatment: take the epidermis and make several pieces of epidermis on the operation table of the epidermal cell enrichment instrument; (2) Digestion: start the epidermal cell enrichment instrument power supply, take the steps (1) The obtained skin sheet is placed in a digestion container, digestive enzyme is added, and the vibrator and the heater are controlled by the control unit to perform dynamic digestion to obtain a cell suspension; (3) filtration: the step (2) The obtained cell suspension is dropped into a filter container for filtration to remove the skin residue, and the lower layer of the cell liquid is used for the dosing; (4) Solution: The cells obtained by filtration are dropped into a mixing container, mixed with a protein gel, and uniformly mixed to complete the solution.
  • the skin taken is a tomographic skin
  • the tomographic skin has a thickness of 0.3-0.6 mm.
  • the medium-thickness skin sheet has a particle size of 1-5 mm for each skin sheet produced.
  • the digestive enzyme is added in a ratio of: 4-6 ml of digestive enzyme per 1-2 cm 2 of the wound skin, and the digestive enzyme is prepared by pancreatin and EDTA, wherein the pancreas
  • the mass concentration of the enzyme in the digestive enzyme is 0.25-0.5%, and the mass concentration of EDTA in the digestive enzyme is 0.01-0.05%; it is allowed to stand for 5-7 minutes (also called static digestion) before dynamic digestion, and then the vibration is started. After 3-7 minutes of dynamic digestion, let stand for another 5-7 minutes, then start the vibrator and cycle 2-5 times at constant temperature.
  • the whole process heater will control the temperature at 36.8-37.5 °C. Scour; the reagent used for cell scouring is either PBS, standard saline or Green's solution until the cells are completely flushed out of the digestion vessel.
  • step (4) The middle and lower cell fluids are added with an inhibitor to form a cell suspension before the protein gel is mixed.
  • the inhibitor is preferably autologous serum or soybean trypsin, and 1-3 drops are added to the autologous serum per 5-15 ml of the lower cell fluid. 15 drops are 1 ml), soy trypsin is added 0.8-3 times of the lower cell fluid.
  • the protein gel is fibrin glue
  • the mixing process is: mixing the cell suspension with the coagulant and fibrin glue respectively.
  • the coagulant and fibrin glue are pre-mixed first, then the cell suspension and coagulant / Fibrin glue is poured into different cylinders of a double-tube syringe, under the same pressure, cell suspension and coagulant / The fibrin glue is simultaneously extruded, and the mixture is uniformly mixed to complete the dosing, the total amount of fibrin glue and the coagulant and the cell suspension.
  • the epidermal cell enrichment meter and the dynamic enrichment method thereof adopt a dynamic digestion method, and on the one hand, the epidermis can be quickly dispersed to obtain a high density epidermal cell suspension, and on the other hand, the digestive enzyme can be used.
  • the concentration and the amount of use are greatly reduced, the digestion is more uniform, the digestion time can be shortened, the cell activity is maintained, the cell loss is reduced, and the differentiated epidermal cells obtained by the final dosing are sprayed on the wound surface, and the epidermal cells pass the inverse on the wound surface. Differentiation forms epidermal stem cells, promotes wound healing, and the skin source is fully and rationally utilized, and the amplification factor is reached. 100-120 times.
  • Figure 1 is a plan view of the epidermal cell enrichment apparatus of the present embodiment
  • FIG. 2 is a schematic perspective view showing the structure of an epidermal cell enrichment apparatus of the present embodiment
  • Figure 3 is a diagram showing the control module of the epidermal cell enrichment meter of the present embodiment
  • Figure 4 is a comparison of staining results at week 2 after application of the cells obtained by the present invention to the back of a nude mouse. Under the microscope)
  • Figure 5 is a comparison of staining results at week 3 after application of the cells obtained by the present invention to the back of a nude mouse. Under the microscope)
  • Figure 6 is a comparison of staining results at week 4 after application of the cells obtained by the present invention to the back of a nude mouse. Under the microscope)
  • Figure 7 shows the enrichment of live epidermal cells obtained by static digestion under a microscope (under a 100-fold phase contrast microscope);
  • Figure 8 shows the enrichment of fibroblasts obtained by dynamic digestion under a microscope (under a 100-fold phase contrast microscope).
  • an epidermal cell enrichment apparatus includes a base 1 and a control panel 2 disposed on the base 1 and a digestion container. 3, filter container 4, mixing container 5, control unit 6, heater 7, vibrator 8, sensor 9 and power supply 10, etc., wherein control panel 2 includes display 21 and buttons 22, in the embodiment, the display screen 21 is displayed by a digital tube, and is mainly used for displaying parameters such as temperature, vibration frequency, cycle time, etc.
  • the button 22 includes a power start button and a vibration open button.
  • Vibrator 8 Installed at the bottom of the digestion vessel 3, the digestion vessel 3 is vibrated at a certain frequency; in the embodiment, the vibrator 8 is a vibration motor, and the vibration motor has a rotational speed range of 1100-2500 rpm. .
  • the vibrator 8 can also be replaced by other similar components, as long as the solution in the digestion vessel 3 produces a certain frequency of vibration or shaking. If a shaker is used instead of the vibrator 8, the speed of the shaker is generally controlled. 60-300rpm.
  • the heater 7 is mounted on the outer surface of the digestion vessel 3 for heating the digestion vessel 3 so that the solution in the digestion vessel 3 is maintained at 36.8-37.5 °C, the sensor 9 Installed on the through hole of the side wall of the digestion vessel 3, the probe of the temperature sensor protrudes into the solution, and the heating temperature of the digestion vessel 3 is monitored in real time, and the temperature sensor 9
  • a variety of temperature sensors can be used in order to accurately sense the temperature of the solution in the digestion vessel 3.
  • the heater 7 and the vibrator 8 are respectively connected to the output of the control unit 6, the control panel 2 and the sensor 9 They are respectively connected to the input terminals of the control unit 6, and the power source 10 supplies power to the control panel 2, the control unit 6, the heater 7, and the vibrator 8, respectively.
  • the above control unit 6 With a microprocessor chip, you can select multiple models of microprocessors, as long as the above control functions can be implemented.
  • the filter container 4 has a 60-mesh filter at the upper end.
  • the above-mentioned digestion container 3, the inlet portion of the filtration container 5 and the mixing container 6, and the control panel 2 are respectively mounted on the upper surface of the base 1 11 on.
  • the capacity of the digestion vessel 3, the filtration vessel 5 and the mixing vessel 6 is generally 10-20 ml.
  • the upper surface 11 of the base is further provided with a console 13 for pre-digestion, shearing and the like.
  • the control unit 6, the power source 10 and the vibrator 8 are mounted on the lower surface 12 of the base 1, respectively, and the vibrator 8 The top is in contact with the bottom of the digestion vessel 3.
  • a method for dynamically enriching epidermal cells comprising pretreatment, digestion, filtration and dosing, the specific steps are:
  • Pretreatment taking the epidermis at the console of the epidermal cell enrichment instrument 13 A plurality of skin sheets are formed on the skin, and the skin is taken as a tomographic skin sheet, wherein the tom skin sheet is a medium-thickness skin sheet having a thickness of 0.3 mm, and the diameter of each of the skin sheets obtained is 1 mm;
  • step (3) Filtration: the cell suspension obtained in step (2) is dropped into a filter container 4 Filtration is carried out to remove the residue of the epidermis, and the cell liquid of the lower layer is used for the liquid preparation.
  • the filter net is 60 mesh;
  • the inhibitor is formed into a cell suspension by adding an inhibitor to the lower cell fluid.
  • the inhibitor is autologous serum, and 1 drop of autologous serum is added per 5 ml of the lower cell fluid (per 15 The drop is 1 ml);
  • the protein gel is fibrin glue, and the mixing process is: mixing the cell suspension with the coagulant and fibrin glue, respectively.
  • the coagulant and fibrin glue can be pre-mixed first, then the cell suspension and coagulant / Fibrin glue is poured into different cylinders of a double-tube syringe, under the same pressure, cell suspension and coagulant / The fibrin glue is simultaneously extruded, and the mixture is uniformly mixed to complete the dosing, the total amount of fibrin glue and the coagulant and the cell suspension.
  • An epidermal cell enrichment apparatus has the same structure as in Example 1, and the main difference is that the filter mesh is 50 mesh.
  • a method for dynamic enrichment of epidermal cells including pretreatment, digestion, filtration and dosing, the specific steps are:
  • Pretreatment taking the epidermis at the console of the epidermal cell enrichment instrument 13 A plurality of skin sheets are formed on the skin; the skin is taken as a tomographic film, and each of the obtained skin sheets has a particle size of 3 mm, and the thickness of the tomographic sheet is 0.5 mm;
  • step (3) Filtration: the cell suspension obtained in step (2) is dropped into a filter container 4 Filtration is performed to remove the residue of the epidermis, and the cell liquid of the lower layer is used for the solution; in the embodiment, the filter is 50 mesh;
  • Dosing dropping the filtered cells into a mixing container 5 Mix with the protein gel and mix well to complete the dosing.
  • an inhibitor is added to the lower cell fluid to form a cell suspension, and the inhibitor soybean trypsin and soybean trypsin are added in equimolar amounts.
  • the protein gel is fibrin glue
  • the mixing process is: mixing the cell suspension with the coagulant and fibrin glue respectively.
  • the coagulant and fibrin glue are pre-mixed, and then the cell suspension and coagulant are added.
  • Fibrin glue is poured into different cylinders of a double-tube syringe, under the same pressure, cell suspension and coagulant / The fibrin glue is simultaneously extruded, and the mixture is uniformly mixed to complete the dosing, the total amount of fibrin glue and the coagulant and the cell suspension.
  • An epidermal cell enrichment apparatus has the same structure as in the first embodiment, and the main difference is that the filter net is 20 mesh.
  • a method for dynamically enriching epidermal cells comprising pretreatment, digestion, filtration and dosing, the specific steps are:
  • Pretreatment taking the epidermis at the console of the epidermal cell enrichment instrument 13 A plurality of skin sheets are formed on the skin; the skin is taken as a sectional skin sheet, and each of the obtained skin sheets has a particle diameter of 5 mm. a medium-thickness skin with a thickness of 0.6 mm of the tomographic skin;
  • 6 ml of digestive enzyme is added per 2 cm 2 of the tomographic skin, and the digestive enzyme is prepared by trypsin and EDTA, wherein the concentration of the trypsin in the digestive enzyme is 0.5%.
  • the concentration of EDTA in the digestive enzyme is 0.05%; it should be allowed to stand for 7 minutes (also called static digestion) before dynamic digestion, then the vibrator 8 is activated, and after dynamic digestion for 7 minutes, it is allowed to stand for another 7 minutes.
  • step (3) Filtration: the cell suspension obtained in step (2) is dropped into a filter container 4 Filtration is performed to remove the residue of the epidermis, and the cell liquid of the lower layer is used for the liquid preparation.
  • the filter net is 20 mesh;
  • an inhibitor is added to the lower cell fluid to form a cell suspension, and the inhibitor is preferably autologous serum, and 3 drops of autologous serum are added per 15 ml of the lower cell fluid (per 15 drops for 1ml).
  • the protein gel is fibrin glue, and the mixing process is: mixing the cell suspension with the coagulant and fibrin glue respectively.
  • the coagulant and fibrin glue are pre-mixed first, then the cell suspension and coagulant / Fibrin glue is poured into different cylinders of a double-tube syringe, under the same pressure, cell suspension and coagulant / The fibrin glue is simultaneously extruded, and the mixture is uniformly mixed to complete the dosing, the total amount of fibrin glue and the coagulant and the cell suspension.
  • the vibrator of the present embodiment functions in the same manner as the shaker for the biological laboratory culture in the first embodiment, and the digesting container 3 is provided.
  • the solution inside generates a certain frequency of vibration or shaking, but the structure and principle of the device are different from those of the biological laboratory culture shaker in Embodiment 1, so the rotational speed of the vibration motor in this embodiment ranges from 1100 to 2500 rpm.
  • the vibration motor speed can be selected as 1100, 1500, 1800, 2300 and 2500 depending on the type of skin selected and the difference in processing volume.
  • the filter container can be used with a mesh size of 30, 40, 50 or 60 to suit the processing and processing purposes.
  • Table 1 is the total epidermal cell acquisition amount in different digestion modes in Example 1, Table 2 The different epidermal cells obtained in the above examples were presented for comparison of wound healing time between groups.
  • the rotation speed (the rotation speed in Table 1 refers to the rotation speed of the biological laboratory culture shaker, model HS-100C) can be under the dynamic condition of 180-240 rpm. A cell acquisition of 1 5 ⁇ 10 6 -24 ⁇ 10 6 was obtained, and the amount of viable epidermal cells obtained was very satisfactory.
  • Table 2 compares the healing time between different epidermal cell presentation groups
  • the gel-like enriched cells obtained in the above examples can be sprayed on the burn wounds, and the cells are evenly distributed on the wound surface, and the distribution of the cells is not less than 4000/cm 2 , because the epidermal cells except the epidermal stem cells are the same. It has the function of proliferation and helps the wound to close. Therefore, in the above scheme, all the tomographic skins are used as the source of epidermal cells.
  • the shaker is used to provide dynamic conditions at a suitable temperature. It is beneficial to screen cells with larger activity, thereby obtaining larger and more cell volume.
  • the concentration and amount of digestive enzymes can be greatly reduced, which not only reduces the cost of the drug, but also reduces the concentration of digestive enzymes.
  • the activity is maintained, and the original cells obtained by the method can also be mixed with other materials.
  • the use of the dynamic digestion technique of the present invention to obtain epidermal cells is a reliable method.
  • the differentiated epidermal cells can be reversely differentiated on the wound surface to form epidermal stem cells, promote wound healing, and the skin source can be fully and rationally utilized, and the amplification factor is achieved.
  • the concentration of trypsin, EDTA, etc. in the digestive enzymes selected is gradually increased to adapt to the processing capacity, and the amount of inhibitor added corresponds to the amount of skin to be treated, such as when the epidermis is When the amount is increased, the trypsin content in the digestive enzyme is 0.3%, and the EDTA content is 0.035%.
  • the rest time is controlled at 6 minutes, the dynamic time is controlled at 4 minutes, the cycle is 4 times, or the digestive enzyme is used.
  • the content of pancreatic enzyme is 0.5%, and the content of EDTA is 0.05%.
  • the rest time is controlled at 5 minutes, the dynamic time is controlled at 7 minutes, and the cycle is 5 times; the rotational speed of dynamic digestion should be controlled appropriately. In the range of too high or low, the amount of final active cells can be affected. Therefore, in the present invention, the rotational speed should be controlled at 180-300 rpm, as shown in Table 1.
  • the amount of active cells obtained is the most ideal; when the skin sheet is a thick skin, the treatment time is reduced relative to the processing time of the medium-thickness skin and the concentration of digestive enzymes, and the filtration target is 20 mesh to strengthen The filtration effect, the trypsin concentration in the digestive enzyme is 0.28%, and the EDTA concentration is 0.02%.
  • the rest time is controlled at 5 minutes
  • the dynamic time is controlled at 4 minutes
  • the cycle is 3 times, or in the digestive enzyme.
  • the concentration of trypsin was 0.38% and the concentration of EDTA was 0.04%.
  • the rest time was controlled at 4 minutes
  • the dynamic time was controlled at 6 minutes, and the cycle was performed 3 times.
  • Figure 4 shows the comparison of the staining results of the wounds after the technical solution of Example 2, wherein Figure 4 is the second week of HE.
  • A is a microcarrier to culture epidermal cells, and it can be seen that the epidermis has formed and is thick, but not uniform, and the stratification is obvious
  • B For the uncultured epidermal cell suspension, a thicker epidermal layer is also formed, which has obvious stratification
  • C is a primary cultured epidermal cell which has also formed a epidermal layer with obvious stratification
  • D For fibrin glue alone, there is no obvious epidermal layer formation, and there are a large number of fibroblasts on the wound surface;
  • Figure 5 shows the histological examination of the animal in the third week, HE staining results:
  • A The epithelial cells are cultured for the microcarriers, and there is a thick but uneven epidermal layer.
  • B is an uncultured epidermal cell suspension with a thicker epidermal layer and is relatively uniform.
  • D is fibrin glue alone, no obvious epidermal layer formation;
  • Figure 6 is the fourth week of animal histological examination, HE staining results: A The epithelial cells are cultured for microcarriers, and a thick but uneven epidermal layer is formed.
  • B is an uncultured epidermal cell suspension with a thicker epidermal layer and is relatively uniform.
  • C is a primary cultured epidermal cell. Thin skin layer, D For fibrin glue alone, no epidermal layer is formed.
  • epidermal stem cells In addition to epidermal stem cells, other cells in the epidermal cells also have a proliferative function, which contributes to wound healing. Therefore, in the above-mentioned scheme provided by the present invention, all the tomographic skin sheets are used as the source of epidermal cells; during the dynamic digestion process
  • the dynamic digestion condition is provided by vibration at a certain temperature, on the one hand, a larger and more cell volume can be obtained, and at the same time, the concentration and usage of the digestive enzyme can be greatly reduced, and the digestion time can be shortened, which is beneficial to the cells.
  • the use of the dynamic digestion method of the present invention to obtain epidermal cells is a reliable method, the differentiated epidermal cells can be reversely differentiated on the wound surface to form epidermal stem cells, promote wound healing, and the skin source is fully obtained. Reasonable use, the amplification factor is reached 100-120 times.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Sustainable Development (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Computer Hardware Design (AREA)
  • Dermatology (AREA)
  • Clinical Laboratory Science (AREA)
  • Physics & Mathematics (AREA)
  • Thermal Sciences (AREA)
  • Mechanical Engineering (AREA)
  • Materials For Medical Uses (AREA)

Abstract

提供了一种表皮细胞富集仪及其动态富集方法，所述表皮细胞富集仪，包括底座，以及设置在底座上的控制面板、消化容器、过滤容器、混合容器、控制单元、加热器、振动器、传感器和电源等，通过动态富集消化方法，得到高密度的表皮细胞悬液，完成配液。

Description

一种表皮细胞富集仪及其动态富集方法 技术领域
本发明涉及一种细胞富集仪及其富集方法，特别是涉及一种表皮细胞富集仪及其动态富集方法。
背景技术
近年来，我国烧伤的发病率为总人口的 2 ‰～ 10 ‰，每年发生烧伤者近千万人，需要住院治疗的达数十万人之多，烧伤治愈后常遗留大量疤痕，对患者的外貌、体型，以及功能、心理方面造成负面影响。
目前，利用有限的皮源对较大面积的烧伤创面进行治疗，主要有两种：
1 、微粒皮技术：将有限的自体皮源使用物理方法进行分散成尽量小的微粒，然后回植到病人的创面上，这种技术可将皮片扩大 10 ～ 20 倍，但使用该物理方法一方面很难将皮片进一步扩大，另一方面表皮贴附率低，难以保证疗效；
2 、 RECELL 技术：因为创面的关闭是通过表皮干细胞的增殖完成， RECELL 技术是将基底层表皮细胞进行静态消化，然后利用喷雾法将表皮细胞喷洒于创面，使用该技术理论上可将皮片扩大 40 ～ 80 倍，表皮干细胞主要存在于基底层中，因此，这种技术主要是利用表皮干细胞来修复创面。采用静态消化主要存在如下缺陷：消化酶与底物之间混合欠佳，消化时间长，且消化不均匀。
有鉴于此，本发明人对此进行研究，专门开发出一种表皮细胞富集仪及其动态富集方法，本案由此产生。
发明内容
本发明的目的是提供一种表皮细胞富集仪及其动态富集方法，采用动态消化方式取得高密度、有活力的表皮细胞，且整个过程消化时间短，消化均匀。
为了实现上述目的，本发明的解决方案是： 一种表皮细胞富集仪，包括底座，以及设置在底座上的控制面板、消化容器、过滤容器、混合容器、控制单元、加热器、振动器、传感器和电源等，其中，控制面板包括显示屏和按键，振动器安装在消化容器的底部，使消化容器产生一定频率的振动；加热器安装在消化容器的外表面上，用于加热消化容器内的液体，温度传感器安装在消化容器上，用于监测消化容器的加热温度，上述加热器、振动器分别与控制单元的输出端相连，控制面板和传感器器分别与控制单元的输入端相连，电源分别为控制面板、控制单元、加热器和振动器供电。
上述消化容器的侧壁上设有一通孔，用于安装温度传感器。
上述过滤容器的上端设有滤网，用于过滤动态消化后的溶液，滤网的目数为 20-60 。
上述消化容器、过滤容器和混合容器的入口部以及控制面板分别安装在底座的上表面上。
上述底座上表面上进一步设有一操作台，用于表皮消化前的剪切等预处理。
所述振动器采用振动电机。
控制单元、电源和振动器分别安装在底座的下表面上，振动器的顶部与消化容器的底部相接触。
一种表皮细胞动态富集方法，包括预处理、消化、过滤和配液，具体步骤为： （ 1 ）预处理：取表皮，在表皮细胞富集仪的操作台上制成若干块表皮片； （ 2 ）消化：启动表皮细胞富集仪电源，取步骤（ 1 ）所得表皮片置于消化容器中，加入消化酶，通过控制单元控制振动器和加热器，进行动态消化，得细胞悬液；（ 3 ）过滤：将步骤（ 2 ）所得细胞悬液滴入过滤容器进行过滤，去除表皮残渣，下层的细胞液用于配液；（ 4 ）配液：将过滤所得细胞液滴入混合容器，与蛋白凝胶混合，混合均匀完成配液。
作为优选，上述步骤（ 1 ）中，所取的表皮为断层皮片，断层皮片为厚度为 0.3-0.6mm 的中厚皮片，制得的每块表皮片的粒径在 1-5mm 。
作为优选，上述步骤（ 2 ）中，消化酶的添加比例为：每 1-2 cm2 断层皮片加 4-6ml 的消化酶，所述消化酶由胰酶和 EDTA 配制而成，其中，胰酶在消化酶中的质量浓度为 0.25-0.5% ， EDTA 在消化酶中的质量浓度为 0.01-0.05% ；动态消化前先静置 5-7 分钟（也可称为静态消化），再启动振动器，进行动态消化 3-7 分钟后，再静置 5-7 分钟，再启动振动器，恒温下循环 2-5 次，整个过程加热器将温度控制在 36.8-37.5 ℃，循环结束后进行细胞冲刷；细胞冲刷采用的试剂为 PBS 、标准生理盐水或格林氏液中的任一种，以细胞完全冲出消化容器为止。
作为优选，在步骤（ 4 ）中，下层细胞液在蛋白凝胶混合前，先添加抑制剂形成细胞悬液，抑制剂优选为自体血清或大豆胰蛋白酶，每 5-15ml 下层细胞液中自体血清添加 1-3 滴（每 15 滴为 1ml ），大豆胰蛋白酶的添加量为下层细胞液的 0.8-3 倍。
作为优选，在步骤（ 4 ）中，蛋白凝胶为纤维蛋白胶，混合过程为：分别将细胞悬液与促凝剂、纤维蛋白胶混合。较优的，促凝剂和纤维蛋白胶先进行预混合，再将细胞悬液和促凝剂 / 纤维蛋白胶灌入双筒注射器的不同筒中，在同样压力作用下，细胞悬液和促凝剂 / 纤维蛋白胶同时挤出，混合均匀完成配液，纤维蛋白胶与促凝剂的总量和细胞悬液等量。
与现有技术相比，上述表皮细胞富集仪及其动态富集方法采用动态消化方式，一方面可以快速离散表皮，得到高密度的表皮细胞悬液，另一方面，可以使消化酶的使用浓度和使用量大幅度降低，消化更均匀，同时可缩短消化时间，有利于细胞活性的保持，减少细胞损耗，最终配液所得的已经分化的表皮细胞喷涂在创面上，表皮细胞在创面通过逆分化形成表皮干细胞，促进伤口愈合，皮源得到充分合理的利用，扩增倍数达到 100-120 倍。
以下结合附图及具体实施例对本发明做进一步详细描述。
附图说明
图 1 为本实施例的表皮细胞富集仪俯视图；
图 2 为本实施例的表皮细胞富集仪立体结构示意图；
图 3 为本实施例的表皮细胞富集仪控制模块图；
图 4 为采用本发明所得细胞应用于裸鼠背部创面后第 2 周的染色结果对照（ 100 倍显微镜下）；
图 5 为采用本发明所得细胞应用于裸鼠背部创面后第 3 周的染色结果对照（ 100 倍显微镜下）；
图 6 为采用本发明所得细胞应用于裸鼠背部创面后第 4 周的染色结果对照（ 100 倍显微镜下）；
图 7 为显微镜下静态消化获得的活的表皮细胞得到富集（ 100 倍相差显微镜下）；
图8为显微镜下动态消化获得的成纤维细胞得到富集（ 100 倍相差显微镜下）。
具体实施方式
实施例 1
如图 1-3 所示，一种表皮细胞富集仪，包括底座 1 ，以及设置在底座 1 上的控制面板 2 、消化容器 3 、过滤容器 4 、混合容器 5 、控制单元 6 、加热器 7 、振动器 8 、传感器 9 和电源 10 等，其中，控制面板 2 包括显示屏 21 和按键 22 ，在本实施例中，上述显示屏 21 采用数码管显示，主要用于显示温度、振动频率、循环时间等参数，按键 22 包括电源启动按键和振动开启按键。振动器 8 安装在消化容器 3 的底部，使消化容器 3 产生一定频率的振动；在本实施例中，所述振动器 8 采用振动电机，振动电机的转速范围为 1100-2500rpm 。振动器 8 也可以采用其他类似部件替代，只要能是消化容器 3 内的溶液产生一定频率的震动或晃动就行，如采用摇床替代振动器 8 ，摇床转速一般控制在 60-300rpm 。加热器 7 安装在消化容器 3 的外表面上，用于加热消化容器 3 ，使消化容器 3 内的溶液保持在 36.8-37.5 ℃之间，传感器 9 安装在消化容器 3 侧壁的通孔上，温度传感器的探头伸入溶液内，实时监测消化容器 3 的加热温度，温度传感器 9 可以采用多种型号的温度传感器，目的在于能精确感应到消化容器 3 内溶液的温度。
上述加热器 7 、振动器 8 分别与控制单元 6 的输出端相连，控制面板 2 和传感器器 9 分别与控制单元 6 的输入端相连，电源 10 分别为控制面板 2 、控制单元 6 、加热器 7 和振动器 8 供电。在本实施例中，上述控制单元 6 采用微处理器芯片，可以选择多个型号的微处理器，只要能实现上述控制功能就行。
过滤容器 4 的上端设有 60 目的过滤网。
上述消化容器 3 、过滤容器 5 和混合容器 6 的入口部以及控制面板 2 分别安装在底座 1 的上表面 11 上。消化容器 3 、过滤容器 5 和混合容器 6 的容量一般为 10-20ml 。
上述底座上表面 11 上进一步设有一操作台 13 ，用于表皮消化前，剪切等预处理。
控制单元 6 、电源 10 和振动器 8 分别安装在底座 1 的下表面 12 上，振动器 8 的顶部与消化容器 3 的底部相接触。
一种表皮细胞动态富集方法，包括预处理、消化、过滤和配液，具体步骤为，
（ 1 ）预处理：取表皮，在表皮细胞富集仪的操作台 13 上制成若干块表皮片，所取的表皮为断层皮片，所述断层皮片为厚度为 0.3mm 的中厚皮片，制得的每块表皮片的粒径在 1mm ；
（ 2 ）消化：启动表皮细胞富集仪电源 10 ，取步骤（ 1 ）所得表皮片置于消化容器 3 中，加入消化酶，通过控制单元 6 控制振动器 8 和加热器 7 ，进行动态消化，得细胞悬液；在本实施例中，每 1 cm2 断层皮片加 4ml 的消化酶，所述消化酶由胰酶和 EDTA 配制而成，其中，胰酶在消化酶中的质量浓度为 0.25% ， EDTA 在消化酶中的质量浓度为 0.01% ；动态消化前要先静置 5 分钟（也可称为静态消化），然后再启动振动器 8 ，进行动态消化 3 分钟后，再静置 5 分钟，再启动振动器 8 ，在 36.8 ℃的恒温下循环 2 次，振动电机的转速为 1100rmp ，循环结束后进行细胞冲刷；细胞冲刷采用的试剂为 PBS ，以细胞完全冲出消化容器 3 为止；
（ 3 ）过滤：将步骤（ 2 ）所得细胞悬液滴入过滤容器 4 进行过滤，去除表皮残渣，下层的细胞液用于配液，在本实施例中，所述过滤网为 60 目；
（ 4 ）配液：将过滤所得细胞液滴入混合容器 5 ，与蛋白凝胶混合，混合均匀完成配液，在本实施例中，下层细胞液中添加抑制剂形成细胞悬液，抑制剂为自体血清，每 5ml 下层细胞液中添加自体血清 1 滴（每 15 滴为 1ml ）；蛋白凝胶为纤维蛋白胶，混合过程为：分别将细胞悬液与促凝剂、纤维蛋白胶混合。可以促凝剂和纤维蛋白胶先进行预混合，再将细胞悬液和促凝剂 / 纤维蛋白胶灌入双筒注射器的不同筒中，在同样压力作用下，细胞悬液和促凝剂 / 纤维蛋白胶同时挤出，混合均匀完成配液，纤维蛋白胶与促凝剂的总量和细胞悬液等量。
实施例 2
一种表皮细胞富集仪其结构同实施例 1 ，主要区别在于过滤网为 50 目。
一种表皮细胞动态富集方法，包括预处理、消化、过滤和配液，具体步骤为：
（ 1 ）预处理：取表皮，在表皮细胞富集仪的操作台 13 上制成若干块表皮片；所取的表皮为断层皮片，制得的每块表皮片的粒径在 3mm ，断层皮片的厚度为 0.5mm 的中厚皮片；
（ 2 ）消化：启动表皮细胞富集仪电源 10 ，取步骤（ 1 ）所得表皮片置于消化容器 3 中，加入消化酶，通过控制单元 6 控制振动器 8 和加热器 7 ，进行动态消化，得细胞悬液；在本实施例中，每 1.5 cm2 断层皮片加 5ml 的消化酶，所述消化酶由胰酶和 EDTA 配制而成，其中，胰酶在消化酶中的质量浓度为 0.4% ， EDTA 在消化酶中的质量浓度为 0.03% ；动态消化前要先静置 6 分钟（也可称为静态消化），然后再启动振动器 8 ，进行动态消化 5 分钟后，再静置 6 分钟，再启动振动器 8 ， 37 ℃恒温下循环 4 次，振动电机的转速为 2000rmp ，循环结束后进行细胞冲刷；细胞冲刷采用的试剂为标准生理盐水，以细胞完全冲出消化容器为止。
（ 3 ）过滤：将步骤（ 2 ）所得细胞悬液滴入过滤容器 4 进行过滤，去除表皮残渣，下层的细胞液用于配液；在本实施例中，所述过滤网为 50 目；
（ 4 ）配液：将过滤所得细胞液滴入混合容器 5 ，与蛋白凝胶混合，混合均匀完成配液。在本实施例中，下层细胞液中添加抑制剂形成细胞悬液，抑制剂大豆胰蛋白酶，大豆胰蛋白酶则以等摩尔量添加。蛋白凝胶为纤维蛋白胶，混合过程为：分别将细胞悬液与促凝剂、纤维蛋白胶混合。较优的，促凝剂和纤维蛋白胶先进行预混合，再将细胞悬液和促凝剂 / 纤维蛋白胶灌入双筒注射器的不同筒中，在同样压力作用下，细胞悬液和促凝剂 / 纤维蛋白胶同时挤出，混合均匀完成配液，纤维蛋白胶与促凝剂的总量和细胞悬液等量。
实施例 3
一种表皮细胞富集仪其结构同实施例 1 ，主要区别在于过滤网为 20 目。
一种表皮细胞动态富集方法，包括预处理、消化、过滤和配液，具体步骤为，
（ 1 ）预处理：取表皮，在表皮细胞富集仪的操作台 13 上制成若干块表皮片；所取的表皮为断层皮片，制得的每块表皮片的粒径在 5mm 。断层皮片的厚度为 0.6mm 的中厚皮片；
（ 2 ）消化：启动表皮细胞富集仪电源，取步骤（ 1 ）所得表皮片置于消化容器中，加入消化酶，通过控制单元 10 控制振动器 8 和加热器 7 ，进行动态消化，得细胞悬液；在本实施例中，每 2 cm2 断层皮片加 6ml 的消化酶，所述消化酶由胰酶和 EDTA 配制而成，其中，胰酶在消化酶中的质量浓度为 0.5% ， EDTA 在消化酶中的质量浓度为 0.05% ；动态消化前要先静置 7 分钟（也可称为静态消化），然后再启动振动器 8 ，进行动态消化 7 分钟后，再静置 7 分钟，再启动振动器 8 ， 37.5 ℃恒温下循环 5 次，振动电机的转速为 2500rmp ，循环结束后进行细胞冲刷，细胞冲刷采用的试剂为格林氏液，以细胞完全冲出消化容器为止；
（ 3 ）过滤：将步骤（ 2 ）所得细胞悬液滴入过滤容器 4 进行过滤，去除表皮残渣，下层的细胞液用于配液，在本实施例中，所述过滤网为 20 目；
（ 4 ）配液：将过滤所得细胞液滴入混合容器 5 ，与蛋白凝胶混合，混合均匀完成配液。在本实施例中，下层细胞液中添加抑制剂形成细胞悬液，抑制剂优选为自体血清，每 15ml 下层细胞液中添加自体血清 3 滴（每 15 滴为 1ml ）。蛋白凝胶为纤维蛋白胶，混合过程为：分别将细胞悬液与促凝剂、纤维蛋白胶混合。促凝剂和纤维蛋白胶先进行预混合，再将细胞悬液和促凝剂 / 纤维蛋白胶灌入双筒注射器的不同筒中，在同样压力作用下，细胞悬液和促凝剂 / 纤维蛋白胶同时挤出，混合均匀完成配液，纤维蛋白胶与促凝剂的总量和细胞悬液等量。
其中涉及到的消化酶以及消化酶添加量、促凝剂、抑制剂、蛋白凝胶等参数的设置可参考实施例 1 中对应的参数，本实施例振动器的作用与实施例 1 中生物实验室培养用 摇床 作用相同，都是使消化容器 3 内的溶液产生一定频率的震动或晃动，但装置结构和原理与实施例 1 中生物实验室培养用 摇床 不同，故本实施例中振动电机的转速范围为 1100-2500rpm ，具体操作中，根据表皮所选种类不同，以及处理量的差别，振动电机的转速可选择为 1100 、 1500 、 1800 、 2300 和 2500 等不同档位，过滤容器可选用目数为 30 、 40 、 50 或 60 ，以适应处理效果和处理目的的需求。
对上述实施例的实施效果进行整体分析。
其中，表 1 为实施例 1 中不同消化方式时的总表皮细胞获取量，表 2 为上述实施例所得不同表皮细胞递呈组间创面愈合时间的比较。
表 1 不同消化方式的总表皮细胞获取量（ P<0.01 ）
消化方式 表皮面积（ cm2 ） 有活力表皮细胞获取量（ 106 ）
静置 / 静态 5 2.42 ± 0.16
转速 60rpm 5 2.35 ± 0.39
转速 120rpm 5 3.26 ± 1.21
转速 180rpm 5 15.04 ± 4.82*
转速 240rpm 5 24.07 ± 7.25*
由表 1 可以看出，当选取同等面积的断层表皮，转速（表 1 中的转速是指生物实验室培养用 摇床 的转速，型号 HS-100C ）在 180-240rpm 时的动态条件下，可以获得 1 5 ×106 -24 ×106的细胞获取量，其活力表皮细胞的获取量非常理想。
表 2 为不同表皮细胞递呈组间创面愈合时间的比较
（ * 表示与纤维蛋白胶组比较， P<0.05 ； ** 表示与纤维蛋白胶比较， P<0.01 ）
组别 天数±标准误差
纤维蛋白胶 15.60 ± 2.98
微载体 8.00 ± 1.23*
悬浮液 6.80 ± 0.86**
静态 7.60 ± 1.08*
采用上述实施例所得的凝胶状的富集细胞喷涂在烧伤创面即可，细胞在创面分布均匀，细胞的分布量不小于 4000 个 /cm2 ，由于表皮细胞中除表皮干细胞外，其他细胞同样具有增殖的功能，有助于创面的关闭，因此，上述方案中，将全部的断层皮片作为表皮细胞来源，在进行动态消化过程中，在适宜温度下采用摇床提供动态条件，一方面可有利于筛选活性较大的细胞，从而获得更大、更多的细胞量，同时，消化酶的使用浓度和使用量也可以大大降低，既降低了药剂成本，消化酶浓度的降低也有利于细胞活性的保持，而本方法所得原细胞也可与其它物料溶合共用。采用本发明中的动态消化技术获得表皮细胞是一种可靠的方法，已经分化的表皮细胞可以在创面通过逆分化，形成表皮干细胞，促进伤口愈合，皮源得到充分合理的利用，扩增倍数达到 120 倍，其中根据处理量的不同，选用的消化酶中胰酶、 EDTA 等的浓度也逐渐增加，以适应处理能力，抑制剂的添加量与待处理的表皮量相对应，如当表皮片的量增加时，消化酶中的胰酶含量为 0.3% 、 EDTA 含量为 0.035% ，对应的消化过程中，静置时间控制在 6 分钟，动态时间控制在 4 分钟，循环 4 次，或者消化酶中的胰酶含量为 0. 5% 、 EDTA 含量为 0.05% ，对应的动态消化过程中，静置时间控制在 5 分钟，动态时间控制在 7 分钟，循环 5 次；动态消化的转速应当控制在适宜的范围内，过高或高低都会影响最终活性细胞的获取量，因而本发明中，转速应控制在 180-300rpm ，如表 1 所示，其活性细胞的获得量最为理想；当采用的表皮片为刃厚皮片时，其处理时间相对于中厚皮片处理时间和消化酶浓度等都要减少，过滤目数为 20 目以强化过滤效果，消化酶中的胰酶浓度为 0.28% 、 EDTA 浓度为 0.02% ，对应的消化过程中，静置时间控制在 5 分钟，动态时间控制在 4 分钟，循环 3 次，或者消化酶中的胰酶浓度为 0. 38% 、 EDTA 浓度为 0.04% ，对应的动态消化过程中，静置时间控制在 4 分钟，动态时间控制在 6 分钟，循环 3 次。
以下以应用于烧伤裸鼠背部创面时的测试结果为例，进行本发明技术方案的具体阐述。
图 4 、图 5 和图 6 为采用实施例 2 技术方案后，创面的染色结果对照，其中图 4 为第二周 HE 染色结果， A 为微载体培养表皮细胞，可见表皮已经形成，并且较厚，但是欠均匀，复层化明显， B 为未经培养的表皮细胞悬液，同样形成较厚的表皮层，有明显的复层化， C 为原代培养表皮细胞也已经形成表皮层，有明显的复层化， D 为单纯纤维蛋白胶，无明显表皮层形成，创面有大量成纤维细胞；图 5 为第三周动物实验组织学检测， HE 染色结果： A 为微载体培养表皮细胞，有较厚的但不均匀的表皮层形成， B 为未经培养的表皮细胞悬液，有较厚的表皮层形成，且比较均匀， C 为原代培养表皮细胞，有较薄的均匀的表皮层形成， D 为单纯纤维蛋白胶，无明显表皮层形成；图 6 为第四周动物实验组织学检测， HE 染色结果： A 为微载体培养表皮细胞，有较厚的但不均匀的表皮层形成， B 为未经培养的表皮细胞悬液，有较厚的表皮层形成，且比较均匀， C 为原代培养表皮细胞，表皮层较薄， D 为单纯纤维蛋白胶，无表皮层形成。
从在图 7 和图 8 中可以明显看出：通过动态消化获得的细胞（图 8 ）明显多于静态消化所获得的细胞（图 7 ），通过台盼蓝染色后，动态消化的细胞仍然维持较高的细胞活力（图 8 ）。
表皮细胞中除表皮干细胞外，其他细胞同样具有增殖的功能，有助于创面愈合，因此，本发明所提供的上述方案中，将全部的断层皮片作为表皮细胞来源；在进行动态消化过程中，在一定温度下采用振动方式提供动态消化条件，一方面可获得更大、更多的细胞量，同时，消化酶的使用浓度和使用量也可以大大降低，同时可缩短消化时间，有利于细胞活性的保持，减少细胞损耗，采用本发明中的动态消化方法获得表皮细胞是一种可靠的方法，已经分化的表皮细胞可以在创面通过逆分化，形成表皮干细胞，促进伤口愈合，皮源得到充分合理的利用，扩增倍数达到 100-120 倍。
上述实施例和图式并非限定本发明的产品形态和式样，任何所属技术领域的普通技术人员对其所做的适当变化或修饰，皆应视为不脱离本发明的专利范畴。

Claims (10)

1.
一种表皮细胞富集仪，其特征在于：包括底座，以及设置在底座上的控制面板、消化容器、过滤容器、混合容器、控制单元、加热器、振动器、传感器和电源，其中，控制面板包括显示屏和按键，振动器安装在消化容器的底部，使消化容器产生一定频率的振动；加热器安装在消化容器的外表面上，用于加热消化容器内的液体，温度传感器安装在消化容器上，用于监测消化容器的加热温度，上述加热器、振动器分别与控制单元的输出端相连，控制面板和传感器器分别与控制单元的输入端相连，电源分别为控制面板、控制单元、加热器和振动器供电。
2.
如权利要求 1 所述的一种表皮细胞富集仪，其特征在于：上述消化容器的侧壁上设有一通孔，用于安装温度传感器。
3.
如权利要求 1 所述的一种表皮细胞富集仪，其特征在于：上述过滤容器的上端设有滤网，用于过滤动态消化后的溶液，滤网的目数为
20-60 。
4.
如权利要求 1 所述的一种表皮细胞富集仪，其特征在于：上述消化容器、过滤容器和混合容器的入口部以及控制面板分别安装在底座的上表面上；上述底座上表面上进一步设有一操作台。
5.
如权利要求 1 所述的一种表皮细胞富集仪，其特征在于：控制单元、电源和振动器分别安装在底座的下表面上，振动器的顶部与消化容器的底部相接触；所述振动器采用振动电机。
6.
根据权利要求3所述的一种涤纶工业丝熔体直纺生产装置，其特征在于：所述纺丝单元与液相增黏釜之间呈'线型'、'星型'或'矩形对称'布置。
一种表皮细胞动态富集方法，其特征在于：包括预处理、消化、过滤和配液，具体步骤为：
（ 1 ）预处理：取表皮，在表皮细胞富集仪的操作台上制成若干块表皮片；
（ 2 ）消化：启动表皮细胞富集仪电源，取步骤（ 1 ）所得表皮片置于消化容器中，加入消化酶，通过控制单元控制振动器和加热器，进行动态消化，得细胞悬液；
（ 3 ）过滤：将步骤（ 2 ）所得细胞悬液滴入过滤容器进行过滤，去除表皮残渣，下层的细胞液用于配液；
（ 4 ）配液：将过滤所得细胞液滴入混合容器，与蛋白凝胶混合，混合均匀完成配液。
7.
如权利要求 6 所述的一种表皮细胞动态富集方法，其特征在于：上述步骤（ 1 ）中，所取的表皮为断层皮片，断层皮片为厚度为 0.3-0.6mm 的中厚皮片，制得的每块表皮片的粒径在 1-5mm 。
8.
如权利要求 7 所述的一种表皮细胞动态富集方法，其特征在于：上述步骤（ 2 ）中， 1-2 cm2 断层皮片加 4-6ml 的消化酶，所述消化酶由胰酶和 EDTA 配制而成，其中，胰酶在消化酶中的质量浓度为 0.25-0.5% ， EDTA 在消化酶中的质量浓度为 0.01-0.05% ；动态消化前先静置 5-7 分钟，再启动振动器，进行动态消化 3-7 分钟后，再静置 5-7 分钟，再启动振动器，恒温下循环 2-5 次，整个过程加热器将温度控制在 36.8-37.5℃，循环结束后进行细胞冲刷；细胞冲刷采用的试剂为PBS 、标准生理盐水或格林氏液中的任一种，以细胞完全冲出消化容器为止。
9.
如权利要求 6 所述的一种表皮细胞动态富集方法，其特征在于：在步骤（ 4 ）中，下层细胞液在蛋白凝胶混合前，先添加抑制剂形成细胞悬液，抑制剂为自体血清或大豆胰蛋白酶，每 5-15ml 下层细胞液中自体血清添加 1-3 滴，大豆胰蛋白酶的添加量为下层细胞液的 0.8-3 倍。
10.
如权利要求 9 所述的一种表皮细胞动态富集方法，其特征在于：在步骤（ 4 ）中，蛋白凝胶为纤维蛋白胶，混合过程为分别将细胞悬液与促凝剂、纤维蛋白胶混合：促凝剂和纤维蛋白胶先进行预混合，再将细胞悬液和促凝剂 / 纤维蛋白胶灌入双筒注射器的不同筒中，在同样压力作用下，细胞悬液和促凝剂 / 纤维蛋白胶同时挤出，混合均匀完成配液，纤维蛋白胶与促凝剂的总量和细胞悬液等量。
PCT/CN2014/076037 2013-11-27 2014-04-23 一种表皮细胞富集仪及其动态富集方法 WO2015078137A1 (zh)

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN201320763903.6 2013-11-27
CN201310610927.2 2013-11-27
CN201310611230.7 2013-11-27
CN201310610927.2A CN103614295B (zh) 2013-11-27 2013-11-27 一种表皮细胞富集仪及其动态富集方法
CN201310611230.7A CN103623001B (zh) 2013-11-27 2013-11-27 表皮细胞的富集应用
CN201310613989.9A CN103642750A (zh) 2013-11-27 2013-11-27 表皮细胞的动态富集方法
CN201320763903.6U CN203625377U (zh) 2013-11-27 2013-11-27 一种表皮细胞富集仪
CN201310613989.9 2013-11-27

Publications (1)

Publication Number Publication Date
WO2015078137A1 true WO2015078137A1 (zh) 2015-06-04

Family

ID=53198284

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2014/076037 WO2015078137A1 (zh) 2013-11-27 2014-04-23 一种表皮细胞富集仪及其动态富集方法

Country Status (1)

Country Link
WO (1) WO2015078137A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9610430B2 (en) 2006-09-11 2017-04-04 Renovacare Sciences Corp. Cell spraying device, method and sprayed cell suspension
US10376658B2 (en) 2011-04-27 2019-08-13 Renovacare Sciences Corp. Device for cell spraying
US11040363B2 (en) 2016-06-14 2021-06-22 Renovacare Sciences Corp. Modular device for cell spraying

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2236288Y (zh) * 1995-09-11 1996-10-02 武汉市第三医院 旋转式医用剪皮机
CN2454382Y (zh) * 2000-11-15 2001-10-17 中国科学院生物物理研究所 一种用于细胞工程和组织工程的滚动培养装置
CN1840647A (zh) * 2006-01-11 2006-10-04 中国人民解放军第三军医大学第三附属医院 一种用于人造皮肤生产及细胞培养的程序换液装置
CN102488930A (zh) * 2011-12-15 2012-06-13 无锡市第三人民医院 一种自异体表皮细胞混合悬液的制备方法
CN102618442A (zh) * 2012-04-06 2012-08-01 中国人民解放军第四军医大学 一种人工皮肤培养装置及其使用方法
CN103614295A (zh) * 2013-11-27 2014-03-05 绍兴振德医用敷料有限公司 一种表皮细胞富集仪及其动态富集方法
CN103623001A (zh) * 2013-11-27 2014-03-12 绍兴振德医用敷料有限公司 表皮细胞的富集应用
CN103642750A (zh) * 2013-11-27 2014-03-19 绍兴振德医用敷料有限公司 表皮细胞的动态富集方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN2236288Y (zh) * 1995-09-11 1996-10-02 武汉市第三医院 旋转式医用剪皮机
CN2454382Y (zh) * 2000-11-15 2001-10-17 中国科学院生物物理研究所 一种用于细胞工程和组织工程的滚动培养装置
CN1840647A (zh) * 2006-01-11 2006-10-04 中国人民解放军第三军医大学第三附属医院 一种用于人造皮肤生产及细胞培养的程序换液装置
CN102488930A (zh) * 2011-12-15 2012-06-13 无锡市第三人民医院 一种自异体表皮细胞混合悬液的制备方法
CN102618442A (zh) * 2012-04-06 2012-08-01 中国人民解放军第四军医大学 一种人工皮肤培养装置及其使用方法
CN103614295A (zh) * 2013-11-27 2014-03-05 绍兴振德医用敷料有限公司 一种表皮细胞富集仪及其动态富集方法
CN103623001A (zh) * 2013-11-27 2014-03-12 绍兴振德医用敷料有限公司 表皮细胞的富集应用
CN103642750A (zh) * 2013-11-27 2014-03-19 绍兴振德医用敷料有限公司 表皮细胞的动态富集方法

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9610430B2 (en) 2006-09-11 2017-04-04 Renovacare Sciences Corp. Cell spraying device, method and sprayed cell suspension
US10376658B2 (en) 2011-04-27 2019-08-13 Renovacare Sciences Corp. Device for cell spraying
US11135380B2 (en) 2011-04-27 2021-10-05 Renovacare Sciences Corp. Device for cell spraying
US11040363B2 (en) 2016-06-14 2021-06-22 Renovacare Sciences Corp. Modular device for cell spraying

Similar Documents

Publication Publication Date Title
Liang et al. Precise intradermal injection of nanofat-derived stromal cells combined with platelet-rich fibrin improves the efficacy of facial skin rejuvenation
WO2015078137A1 (zh) 一种表皮细胞富集仪及其动态富集方法
Ghazanfari et al. Effects of cyclic stretch on proliferation of mesenchymal stem cells and their differentiation to smooth muscle cells
CN103142447A (zh) 一种表皮干细胞活性成分及含有该活性成分的美容制剂
CN103623001B (zh) 表皮细胞的富集应用
Tang et al. Roles of pulmonary telocytes in airway epithelia to benefit experimental acute lung injury through production of telocyte-driven mediators and exosomes
Yang et al. Requirement for serum in medium supplemented with insulin-transferrin-selenium for hydrodynamic cultivation of engineered cartilage
Rattanasuwan et al. Platelet-rich plasma stimulated proliferation, migration, and attachment of cultured periodontal ligament cells
Lee et al. Wound healing effects of paste type acellular dermal matrix subcutaneous injection
Chu et al. Effects of transplanted adipose derived stem cells on the expressions of α‑SMA and DCN in fibroblasts of hypertrophic scar tissues in rabbit ears
Deshpande et al. Silk fibroin and ceramic scaffolds: Comparative in vitro studies for bone regeneration
Zhang et al. Efficient scarless skin regeneration enabled by loading micronized amnion in a bioinspired adhesive wound dressing
Falquet et al. Healthy and patient Type 2 innate lymphoid cells are differently affected by in vitro culture conditions
Li et al. Sandwich biomimetic scaffold based tendon stem/progenitor cell alignment in a 3d microenvironment for functional tendon regeneration
Lu et al. Injectable 3D‐Printed Porous Scaffolds for Adipose Stem Cell Delivery and Endometrial Regeneration
Huang et al. Calcitonin gene-related peptide-induced calcium alginate gel combined with adipose-derived stem cells differentiating to osteoblasts
CN103642750A (zh) 表皮细胞的动态富集方法
Yang et al. Effects of bone marrow stromal cell-conditioned medium on primary cultures of peripheral nerve tissues and cells
CN103614295B (zh) 一种表皮细胞富集仪及其动态富集方法
Hamel et al. Breast Cancer-Stromal Interactions: Adipose-Derived Stromal/Stem Cell Age and Cancer Subtype Mediated Remodeling
CN113652398A (zh) 增强间充质干细胞外泌体黏膜修复功效的方法及复合物
Zhang et al. Edaravone combined with Schwann cell transplantation may repair spinal cord injury in rats
Nouri et al. Comparison of full rotation and reciprocating movements in regaining apical patency during endodontic retreatment
CN111265550A (zh) 一种修复损伤组织的干细胞因子脂质体及其制备方法
CN110193236A (zh) 一种高化学稳定型滤料的制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14865571

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 14865571

Country of ref document: EP

Kind code of ref document: A1