CN114686419A

CN114686419A - Method and device for isolated culture of human epidermal stem cells

Info

Publication number: CN114686419A
Application number: CN202210289257.8A
Authority: CN
Inventors: 陈志超; 赵振民; 张心灵; 李义
Original assignee: Beijing Zhongke Jianlan Group Co ltd; Peking University Third Hospital Peking University Third Clinical Medical College
Current assignee: Beijing Zhongke Jianlan Group Co ltd; Peking University Third Hospital Peking University Third Clinical Medical College
Priority date: 2022-03-23
Filing date: 2022-03-23
Publication date: 2022-07-01
Anticipated expiration: 2042-03-23
Also published as: CN114686419B

Abstract

The invention discloses a separation culture method and a separation culture device for human epidermal stem cells, and relates to the technical field of stem cell culture. The separation culture method comprises the steps of material selection, pretreatment, epidermis trimming and filtering, enzyme digestion blowing, filtration centrifugation, single cell suspension preparation, constant temperature incubation culture and collection of foreskin of a child, and then cleaning, disinfection and removal of subcutaneous tissue.

Description

Method and device for isolated culture of human epidermal stem cells

Technical Field

The invention relates to the technical field of stem cell culture, in particular to a method and a device for separating and culturing human epidermal stem cells.

Background

In recent years, people construct various tissue engineering skins according to the physiological structure of the skin, and are expected to solve the problems of skin source shortage and prognosis scar, wherein seed cells of the tissue engineering skins are mainly epidermal stem cells, fibroblasts and the like. Therefore, isolation and culture of epidermal stem cells are a problem to be faced. In previous reports, the epidermal layer of the human epidermal stem cell is usually separated by digesting with neutral protease overnight and then digesting with 0.25% trypsin at 37 ℃ for 10-15 min, but the defects of poor cell viability, low yield and the like are found in practical application.

The separation culture method and the device of the human epidermal stem cells in the prior art have the following technical problems: the manual epidermis trimming efficiency is low, and the temperature in the enzyme digestion process cannot be kept constant; the integrated device for trimming, enzyme digestion and filtration centrifugation of the epidermis is lacked, and the efficiency of separation culture is reduced.

Disclosure of Invention

The invention aims to provide a separation culture method and a separation culture device for human epidermal stem cells, which are used for solving the problems that the efficiency of manually trimming epidermis is low and the temperature in the enzyme digestion process cannot be kept constant in the prior art; lacks an integrated device for trimming epidermis, enzyme digestion and filtration centrifugation, and reduces the efficiency of separation culture.

The purpose of the invention can be realized by the following technical scheme:

the method for separating and culturing the human epidermal stem cells comprises the following steps:

step one, material selection and pretreatment: selecting foreskin of children of 2-6 years old, soaking for 5-10 min by iodophor, deiodinating by alcohol, removing subcutaneous tissue under aseptic condition, trimming residual tissue to obtain skin pieces of 1cm multiplied by 1cm, digesting the skin pieces for 12-14 hours at 3-5 ℃ in dark by using Dispase enzyme, and separating to obtain epidermis and dermis;

step two, skin trimming and filtering: the epidermis passes through a separation culture device of human epidermal stem cells, and the trimmed epidermis is obtained by trimming and filtering;

step three, enzymatic digestion and blowing: digesting the trimmed epidermis by 0.25 wt% of trypsin at 37 ℃ for 10-15 min; adding fetal calf serum to stop digestion, and blowing to obtain blow liquid; wherein, the trypsin contains 0.04 wt% of EDTA;

step four, filtering and centrifuging: after filtering the blow-beating liquid, centrifugally separating at 1400-1600 rpm for 5-8 min, and standing for layering to obtain supernatant on the upper layer and epidermal stem cell liquid on the lower layer;

step five, preparing single cell suspension: observing cells in epidermal stem cell fluid by inverted microscope, counting, determining the amount of culture medium for suspending cells, adding appropriate amount of K-SFM culture medium, suspending cells, counting, and adjusting cell concentration to 5 × 10⁵Preparing single cell suspension per mL;

step six, incubation culture at constant temperature: inoculating the single cell suspension into a six-well plate previously plated with type IV collagen, incubating overnight at constant temperature, changing the culture medium after 24 hours, and placing at 37 deg.C and 5% CO₂And (4) continuing culturing in the environment, changing the culture solution every 2-3 days, and carrying out passage when the cell growth is fused to 70%.

Further, the specific process of the step two is as follows: the epidermis after adding along the feeder hopper and separating with the corium layer starts the first motor of epidermis pruning intracavity, and first motor drives through first shaft coupling and prunes the axle, prunes the blade rotation, and a plurality of pruning blades prune the cutting to the epidermis, and the air that blows out from blowing the incasement makes the epidermis unordered float in epidermis pruning intracavity, reaches the pruning epidermis that filter plate filtration pore size required and filters the back epidermis and filter the orderly digester that falls into of passageway through the filter.

Further, the specific process of the third step is as follows: adding 0.25 wt% of trypsin from a liquid inlet hopper, driving a cam to rotate by an electric motor, pushing a push-pull head by the cam in a reciprocating manner, pushing the push-pull rod by the push-pull head to extrude the inside of the liquid injection cavity, pushing the trypsin into the liquid injection pipe under pressure, and uniformly dripping the trypsin into a digestion tank; the PTC heating element is electrified by a lead and then heated, the PLC is in communication connection with the temperature sensor, the PLC is electrically connected with the PTC heating element, when the temperature sensor detects that the temperature reaches 37 ℃, a control signal is sent to the PLC, and the PLC closes a conducting circuit of the PTC heating element, so that the temperature of liquid in the digestion tank is kept at 37 ℃;

after digestion is finished, adding fetal calf serum from the liquid inlet hopper, driving the cam to rotate by the electric motor, pushing the push-pull head by the cam in a reciprocating manner, pushing the push-pull rod by the push-pull head to extrude the push-pull rod into the liquid inlet cavity, pushing the fetal calf serum into the liquid inlet pipe under pressure, and uniformly dripping the fetal calf serum into the digestion tank; after the fetal calf serum is added, slowly pushing air through the liquid injection pipe to blow and beat cells to obtain a blow-beating liquid; the blow-beating liquid is discharged into the centrifugal tube through the one-way valve and the liquid discharge tube.

The separation culture device for the human epidermal stem cells comprises a separation culture box, wherein an epidermal trimming cavity, a blowing and filtering cavity, a digestion blowing cavity and a centrifugal separation cavity are sequentially arranged in the separation culture box from top to bottom; a first motor is arranged above the epidermis trimming cavity, the first motor is connected with a trimming shaft extending into the epidermis trimming cavity through a first coupler, a plurality of trimming blades are arranged on the periphery of the trimming shaft, a filter plate is arranged between the epidermis trimming cavity and the purging filter cavity, a stabilizing plate is arranged between the filter plate and the inner wall of the separation culture box, and a purging box facing the center of the epidermis trimming cavity is arranged on the stabilizing plate; a plurality of partition plates communicated with the filter plates are arranged in the sweeping filter cavity, a filter passage is formed between every two adjacent partition plates, and a feed hopper is arranged at the top of the skin trimming cavity.

Furthermore, a plurality of air blowers are arranged in the blowing filter cavity, a blowing pipe penetrating through the stabilizing plate is arranged at the bottom of the blowing box and communicated with the air blowers through connecting flanges, a plurality of blowing heads are uniformly distributed on the upper surface of the blowing box, and a plurality of blowing holes are distributed at the end parts of the blowing heads.

Furthermore, a digestion fixing frame is arranged in the center of the digestion blowing cavity, constant-temperature heating mechanisms are arranged on two sides of the digestion fixing frame, a digestion tank is fixed in the digestion fixing frame, and vent holes are uniformly distributed on the wall of the digestion fixing frame; an enzyme injection mechanism and a fetal calf serum injection mechanism are respectively arranged at the positions of the two sides of the outer wall of the separation culture box, which are positioned in the digestion blow-beating cavity.

Further, constant temperature heating mechanism includes constant temperature heating cabinet, PLC controller and temperature sensor, and temperature sensor locates the outer wall of the fixed frame of digestion, and the constant temperature heating cabinet includes both sides open-ended constant temperature heating frame, and a plurality of aluminum pipes of constant temperature heating frame inner chamber equidistance distribution are connected with the heat pipe between the inner wall of aluminum pipe and constant temperature heating frame, between the adjacent aluminum pipe, have put the PTC heating element in the aluminum pipe, and the wall portion of constant temperature heating frame runs through and is equipped with the heat transfer hole.

Further, the center of the bottom of the digestion fixing frame is connected with a second motor through a second coupler, and a motor fixing frame is arranged on the periphery of the second motor; the bottom of the digestion tank is connected with a plurality of one-way valves which penetrate through the fixed digestion frame, and the bottoms of the one-way valves are connected with a liquid discharge pipe which extends downwards.

Further, enzyme injection mechanism and fetal calf serum injection mechanism's structure is the same, all include the fixed plate, annotate the liquid chamber and annotate the liquid pipe, the fixed plate, annotate the outer wall connection of liquid chamber and separable incubator, the one end of annotating the liquid pipe and annotate liquid chamber intercommunication, other end slope downwardly extending to the top of digester, the top of annotating the liquid chamber is equipped with into the liquid fill, it keeps away from separable incubator outer wall adaptation and is equipped with the push-and-pull rod to annotate the liquid intracavity, the end connection of push-and-pull rod has the push-and-pull head of stretching out the liquid chamber, the outside of push-and-pull head is equipped with the cam, the cam is rotatory through the electric motor drive of arranging in on the fixed plate.

Furthermore, a centrifugal separation mechanism is arranged in the centrifugal separation cavity and comprises a third motor, a vibration damping box and a centrifugal separation box, the third motor is arranged at the bottom of the separation culture box, the vibration damping box and the centrifugal separation box are arranged at the bottom in the centrifugal separation cavity, the centrifugal separation box is arranged above the vibration damping box, the third motor is connected with a centrifugal shaft extending into the vibration damping box through a third coupling, and a plurality of vibration damping springs are arranged above the inner cavity of the vibration damping box; the upper surface annular array of centrifugal separation case distributes a plurality of centrifuging tubes that correspond with the fluid-discharge tube, and the inner chamber top of centrifuging tube is equipped with the screen cloth, and the bottom of centrifuging tube is connected with the collection liquid chamber through dividing the liquid pipe.

The invention has the following beneficial effects:

1. the separation culture method of human epidermal stem cells comprises the steps of collecting foreskins of children, cleaning, disinfecting and removing subcutaneous tissues, finishing the processes of cuticle trimming and filtering, enzyme digestion and blowing, filtering and centrifuging through a separation culture device, wherein the separation culture device can improve the cuticle trimming efficiency, keep the temperature constant in the enzyme digestion process, automatically collect epidermal stem cell liquid, and facilitate the subsequent single cell suspension preparation and constant temperature incubation culture processes.

2. Human epidermis stem cell's isolated culture device, the first motor in epidermis pruning intracavity starts the back, adds the epidermis after with the corium layer separation along the feeder hopper, and first motor drives through first shaft coupling and prunes the axle, prunes the blade rotation, and a plurality of pruning blades prune the epidermis and cut, and the air that blows off from sweeping the incasement makes the epidermis float at the epidermis in disorder in epidermis pruning intracavity, increases the contact probability of pruning blade and epidermis, improves the pruning efficiency of epidermis. Due to the structural arrangement in the epidermis trimming cavity, the epidermis separated from the dermis layer is high in trimming efficiency and uniform in size after trimming.

3. The constant temperature heating mechanism is arranged, after current is introduced into the plurality of PTC heating elements, the PTC heating elements are heated to generate heat, the heat is diffused through the heat transfer holes after being conducted through the aluminum pipes and the heat conduction pipes, so that the temperature in the digestion blowing cavity is raised, the digestion tank absorbs heat to raise the temperature of the epidermis and the enzyme liquid to 37 ℃, the liquid temperature in the digestion tank is kept at 37 ℃ by combining the intelligent control of the PLC and the temperature sensor, and the stability of the enzyme digestion process is kept.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 is a flow chart of a method for isolating and culturing human epidermal stem cells in an embodiment of the present invention;

FIG. 2 is a schematic structural diagram of an isolated culture apparatus for human epidermal stem cells in an embodiment of the present invention;

FIG. 3 is an enlarged view of a portion of the invention at A in FIG. 2;

FIG. 4 is a schematic view of an assembly structure of the purge tank, the blast pipe and the connecting flange according to an embodiment of the present invention;

FIG. 5 is a schematic view of an assembly structure of the digestion fixing frame, the digestion tank and the second motor in the embodiment of the invention;

FIG. 6 is a schematic structural view of a thermostatic heating chamber in an embodiment of the present invention;

FIG. 7 is a schematic structural view of a centrifugal separation mechanism according to an embodiment of the present invention;

FIG. 8 is a cross-sectional view of a centrifugal separation mechanism in an embodiment of the present invention;

FIG. 9 is a schematic structural view of an enzyme injection mechanism or a fetal bovine serum injection mechanism according to an embodiment of the present invention.

In the figure: 10. separating the incubator; 20. a skin trimming chamber; 21. a first motor; 22. a first coupling; 23. trimming the shaft; 24. trimming the leaves; 25. a filter plate; 26. a stabilizing plate; 27. purging the tank; 30. purging the filter cavity; 31. a partition plate; 32. a filtration channel; 33. a blower; 34. a blast pipe; 35. a connecting flange; 36. a sweeping head; 37. a purge hole; 38. a feed hopper; 40. digesting and beating the cavity; 41. a digestion fixing frame; 42. a digestion tank; 43. a vent hole; 44. a second coupling; 45. a second motor; 46. a motor fixing frame; 47. a one-way valve; 48. a liquid discharge pipe; 50. a centrifugal separation chamber; 51. a third motor; 52. a vibration damping box; 53. a centrifugal separation tank; 54. a third coupling; 55. a centrifugal shaft; 56. a vibration damping spring; 57. centrifuging the tube; 58. a screen mesh; 59. a liquid separating pipe; 60. a constant temperature heating mechanism; 61. a constant temperature heating box; 62. a PLC controller; 63. a temperature sensor; 64. a constant temperature heating frame; 65. an aluminum tube; 66. a heat conducting pipe; 67. a PTC heating element; 68. a heat transfer aperture; 69. a liquid collection cavity; 70. an enzyme injection mechanism; 71. a fixing plate; 72. a liquid injection chamber; 73. a liquid injection pipe; 74. a liquid inlet hopper; 75. a push-pull rod; 76. pushing the pull head; 77. a cam; 78. an electric motor; 80. fetal bovine serum infusion mechanism.

Detailed Description

The technical solutions of the present invention will be described below clearly and completely in conjunction with the embodiments, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

As shown in fig. 1, the present embodiment provides a method for isolated culture of human epidermal stem cells, comprising the following steps:

step three, enzymatic digestion and blowing: digesting the trimmed epidermis by 0.25 wt% of trypsin at 37 ℃ for 10-15 min; adding fetal calf serum to stop digestion, and blowing to obtain blow liquid; wherein the trypsin contains 0.04 wt% of EDTA.

Digesting with 0.25% trypsin for about 5min during subculture, stopping digestion, gently blowing adherent cells with a pipette, collecting cell suspension, centrifuging at 1000rpm for 5min, discarding supernatant, resuspending cells, and counting. According to l: passage at 3 ratio, 5% CO at 37 ℃₂Culturing in a constant temperature incubator. And regularly observing the cell morphology and the growth condition under an inverted microscope, and changing the liquid according to the growth condition.

The method for separating and culturing the human epidermal stem cells comprises the steps of material selection, pretreatment, epidermal trimming and filtering, enzyme digestion blowing, filtering and centrifuging, single cell suspension preparation and constant-temperature incubation culture, and after foreskin of a child is collected, subcutaneous tissues are cleaned, disinfected and removed.

Referring to fig. 2-9, the specific process of step two is: the epidermis separated from the dermis layer is added along the feed hopper 38, the first motor 21 in the epidermis trimming cavity 20 is started, the first motor 21 drives the trimming shaft 23 and the trimming blades 24 to rotate through the first coupler 22, the plurality of trimming blades 24 trim and cut the epidermis, the air blown out from the blowing box 27 enables the epidermis to float in the epidermis trimming cavity 20 in a disordered manner, and the trimmed epidermis meeting the size requirement of the filtering holes of the filtering plate 25 is filtered by the filtering plate 25 and then orderly falls into the digestion tank 42 through the filtering channel 32.

The concrete process of the third step is as follows: 0.25 wt% of trypsin is added from the liquid inlet hopper 74, the electric motor 78 drives the cam 77 to rotate, the cam 77 reciprocally pushes the push-pull head 76, the push-pull head 76 pushes the push-pull rod 75 to extrude into the liquid injection cavity 72, and the trypsin is pushed into the liquid injection pipe 73 under pressure and uniformly drops into the digestion tank 42; the PTC heating piece 67 is electrified by a lead to heat and rise temperature, the PLC 62 is in communication connection with the temperature sensor 63, the PLC 62 is electrically connected with the PTC heating piece 67, when the temperature sensor 63 detects that the temperature reaches 37 ℃, a control signal is sent to the PLC 62, and the PLC 62 closes a conducting circuit of the PTC heating piece 67, so that the temperature of liquid in the digestion tank 42 is kept at 37 ℃;

after digestion is finished, the fetal calf serum is added from the liquid inlet hopper 74, the electric motor 78 drives the cam 77 to rotate, the cam 77 reciprocally pushes the push-pull head 76, the push-pull head 76 pushes the push-pull rod 75 to extrude into the liquid inlet cavity 72, and the fetal calf serum is pushed into the liquid inlet pipe 73 under pressure and uniformly drips into the digestion tank 42; after the fetal calf serum is added, slowly pushing air through the liquid injection pipe 73 to blow and beat cells to obtain a blow-beating liquid; the blow-off liquid is discharged into a centrifugal tube 57 through a check valve 47 and a liquid discharge tube 48.

The concrete process of the fourth step is as follows: the multiple centrifuge tubes 57 filter and remove impurities from the blowing and beating liquid flowing in from the liquid discharge tube 48 through the screen 58, the third motor 51 drives the centrifuge shaft 55 to rotate through the third coupler 54, the centrifuge shaft 55 drives the vibration damping box 52 and the centrifugal separation box 53 to rotate, so that the blowing and beating liquid in the centrifuge tubes 57 is centrifugally separated, the supernatant on the upper layer and the epidermal stem cell liquid on the lower layer are obtained by standing and layering, and the epidermal stem cell liquid enters the liquid collecting cavity 69 through the liquid separating tube 59.

Example 2

As shown in fig. 2-3, the present embodiment provides a separation and culture apparatus for human epidermal stem cells, which is used for performing processes of epidermal pruning, filtration, enzymatic digestion, beating, filtration and centrifugation in a separation and culture process of human epidermal stem cells, and includes a separation and culture box 10, wherein the separation and culture box 10 is provided with an epidermal pruning chamber 20, a purging and filtration chamber 30, a digestion beating chamber 40 and a centrifugation and separation chamber 50 sequentially from top to bottom. The top in chamber 20 is pruned to the epidermis is equipped with first motor 21, and first motor 21 is connected with the pruning axle 23 that stretches into in the chamber 20 is pruned to the epidermis through first shaft coupling 22, and the periphery of pruning axle 23 is equipped with a plurality of pruning blades 24, and chamber 20 is pruned to the epidermis and sweep and be equipped with filter 25 between the filter 30, is equipped with steadying plate 26 between the inner wall of filter 25 and separation culture case 10, is equipped with on the steadying plate 26 towards the case 27 that sweeps in chamber 20 center is pruned to the epidermis. A plurality of partition plates 31 communicated with the filter plates 25 are arranged in the sweeping filter cavity 30, a filter channel 32 is formed between the adjacent partition plates 31, and a feed hopper 38 is arranged at the top of the skin trimming cavity 20. Wherein, the filter pore size of filter 25 is 1 ~ 2mm, and firm board 26 is by being close to the slope of the inner wall side of separation incubator 10 to keeping away from the slope of the inner wall side of separation incubator 10 and setting up downwards, and the interval between the adjacent division board 31 is 3 ~ 5 mm.

In the isolated culture device for human epidermal stem cells, after the first motor 21 in the epidermal trimming chamber 20 is started, the epidermis separated from the dermis layer is added along the feed hopper 38, the first motor 21 drives the trimming shaft 23 and the trimming blades 24 to rotate through the first coupler 22, the plurality of trimming blades 24 trim and cut the epidermis, the air blown out from the purging box 27 enables the epidermis to float disorderly in the epidermal trimming chamber 20, the contact probability of the trimming blades 24 and the epidermis is increased, the trimming efficiency of the epidermis is improved, and the trimmed epidermis meeting the size requirement of the filter pores of the filter plate 25 is orderly filtered by the filter plate 25 and then falls into the purging filter chamber 30 through the filter channel 32. The structure in the epidermis trimming cavity 20 is arranged, so that the epidermis separated from the dermis layer has high trimming efficiency and uniform size after trimming.

As shown in fig. 3-4, a plurality of blowers 33 are disposed in the purging filter cavity 30, a blast pipe 34 penetrating through the stabilizing plate 26 is disposed at the bottom of the purging box 27, the blast pipe 34 is communicated with the blowers 33 through a connecting flange 35, a plurality of purging heads 36 are uniformly distributed on the upper surface of the purging box 27, and a plurality of purging holes 37 are distributed at the end of the purging heads 36. After the blower 33 blows air into the blowing box 27 through the blower pipe 34, the air blows dense air in different directions through the blowing holes 37 on the plurality of blowing heads 36, so that the epidermis floats in disorder in the epidermis trimming cavity 20, and the contact area between the epidermis trimming cavity and the trimming blades 24 is increased.

As shown in fig. 2 and 5-6, a digestion fixing frame 41 is arranged in the center of the digestion blowing cavity 40, a constant temperature heating mechanism 60 is arranged on both sides of the digestion fixing frame 41, a digestion tank 42 is fixed in the digestion fixing frame 41, and vent holes 43 are uniformly distributed on the wall of the digestion fixing frame 41. An enzyme injection mechanism 70 and a fetal calf serum injection mechanism 80 are respectively arranged at the two sides of the outer wall of the separation culture box 10, which are positioned in the digestion and blow-up cavity 40.

The digestion tank 42 fixed in the digestion fixing frame 41 is convenient for receiving the fallen trimmed epidermis, the digestion after the enzyme is added, and the blowing after the fetal calf serum is added. The enzyme injection mechanism 70 is capable of adding an enzyme to the digestion tank 42, and the fetal calf serum injection mechanism 80 is capable of adding fetal calf serum to the digestion tank 42.

The constant temperature heating mechanism 60 comprises a constant temperature heating box 61, a PLC 62 and a temperature sensor 63, the temperature sensor 63 is arranged on the outer wall of the digestion fixing frame 41, the constant temperature heating box 61 comprises a constant temperature heating frame 64 with openings at two sides, a plurality of aluminum pipes 65 are distributed in the inner cavity of the constant temperature heating frame 64 at equal intervals, a heat conduction pipe 66 is connected between each aluminum pipe 65 and the inner wall of the constant temperature heating frame 64 and between adjacent aluminum pipes 65, a PTC heating element 67 is inserted in each aluminum pipe 65, and a heat conduction hole 68 is formed in the wall of the constant temperature heating frame 64 in a penetrating mode.

PTC heating element 67 is heated up by generating heat after the wire circular telegram, and PLC controller 62 and temperature sensor 63 communication connection, and PLC controller 62 is connected with PTC heating element 67 electricity, and when temperature sensor 63 detected the temperature and reached 37 ℃, send control signal to PLC controller 62, and PLC controller 62 closes the conducting circuit of PTC heating element 67, and intelligent control makes the liquid temperature in the digestion tank 42 keep 37 ℃.

When current is introduced into the plurality of PTC heating elements 67, the PTC heating elements 67 are heated to generate heat, the heat is conducted through the aluminum pipe 65 and the heat conduction pipe 66 and then diffused through the heat conduction holes 68, so that the temperature in the digestion blow-beating cavity 40 is increased, and the digestion tank 42 absorbs heat and is heated to raise the temperature of the epidermis and the enzyme solution to 37 ℃. The liquid temperature in the digestion tank 42 is kept at 37 ℃ by combining the intelligent control of the PLC 62 and the temperature sensor 63, and the stability in the enzyme digestion process is kept.

The center of the bottom of the digestion fixing frame 41 is connected with a second motor 45 through a second coupling 44, and a motor fixing frame 46 is arranged on the periphery of the second motor 45. The bottom of the digestion tank 42 is connected with a plurality of one-way valves 47 penetrating through the digestion fixing frame 41, and the bottom of the plurality of one-way valves 47 is connected with a drain pipe 48 extending downwards. The second motor 45 drives the digestion fixing frame 41 to rotate through the second coupling 44, and the digestion fixing frame 41 drives the digestion tank 42 to rotate, so that the digestion blowing efficiency is improved; the blowing liquid after digestion blowing is discharged through a one-way valve 47 and a liquid discharge pipe 48.

As shown in fig. 2 and 9, the enzyme injection mechanism 70 and the fetal calf serum injection mechanism 80 have the same structure, and both comprise a fixing plate 71, an injection cavity 72 and an injection pipe 73, the fixing plate 71 and the injection cavity 72 are connected with the outer wall of the separation incubator 10, one end of the injection pipe 73 is communicated with the injection cavity 72, the other end of the injection pipe 73 extends obliquely downwards to the upper part of the digestion tank 42, the top of the injection cavity 72 is provided with a liquid inlet hopper 74, a push-pull rod 75 is arranged in the injection cavity 72 and away from the outer wall of the separation incubator 10 in a matching way, the end part of the push-pull rod 75 is connected with a push-pull head 76 extending out of the injection cavity 72, the outer side of the push-pull head 76 is provided with a cam 77, and the cam 77 is driven to rotate by an electric motor 78 arranged on the fixing plate 71.

When the electric motor 78 drives the cam 77 to rotate, the cam 77 can reciprocally push the push-pull head 76, the push-pull head 76 pushes the push-pull rod 75 to extrude into the liquid injection cavity 72, and after the enzyme or the fetal calf serum is added from the liquid inlet hopper 74, the enzyme or the fetal calf serum is pushed into the liquid injection pipe 73 under pressure and uniformly drops into the digestion tank 42.

As shown in fig. 2 and 7-8, a centrifugal separation mechanism is arranged in the centrifugal separation chamber 50, the centrifugal separation mechanism includes a third motor 51, a vibration damping box 52 and a centrifugal separation box 53, the third motor 51 is arranged at the bottom of the separation incubator 10, the vibration damping box 52 and the centrifugal separation box 53 are arranged at the bottom of the centrifugal separation chamber 50, the centrifugal separation box 53 is arranged above the vibration damping box 52, the third motor 51 is connected with a centrifugal shaft 55 extending into the vibration damping box 52 through a third coupling 54, and a plurality of vibration damping springs 56 are arranged above the inner cavity of the vibration damping box 52. A plurality of centrifuging tubes 57 corresponding to fluid-discharge tube 48 are distributed to the annular array of upper surface of centrifugal separation case 53, and the inner chamber top of centrifuging tube 57 is equipped with screen cloth 58, and the bottom of centrifuging tube 57 is connected with liquid collecting cavity 69 through liquid-separating tube 59. Wherein, the mesh size of the screen 58 is 200-300 meshes.

The third motor 51 drives the centrifugal shaft 55 to rotate through the third coupler 54, the centrifugal shaft 55 drives the slow vibration box 52 and the centrifugal separation box 53 to rotate, the slow vibration spring 56 can relieve vibration generated by the centrifugal separation box 53 in the rotating process, the blow-beating liquid flowing in from the liquid discharge pipe 48 is filtered and decontaminated through the screen 58 and then centrifugally separated by the centrifugal tubes 57, supernatant on the upper layer and epidermis stem cell liquid on the lower layer are obtained after layering, and the epidermis stem cell liquid enters the liquid collection cavity 69 through the liquid distribution pipe 59 to be subjected to the next treatment process.

Examples of the experiments

Immunochemical staining is carried out on the epidermal stem cells separated and cultured in the example 1, the cells are pasted on a cell, PBS is washed, 4% paraformaldehyde is fixed for 20min, immunocytochemical staining detection of beta 1 integrin and CKl9 is carried out by adopting an SP method according to the using instruction steps of the reagent, DAB color development is carried out after primary antibody and secondary antibody are dripped, counterstaining, dehydration, transparency and mounting are carried out, and the observation is carried out under the mirror. Both the epidermal stem cell marker β 1 integrin and CKl9 reacted positively.

The foregoing is merely exemplary and illustrative of the present invention and various modifications, additions and substitutions may be made by those skilled in the art to the specific embodiments described without departing from the scope of the invention as defined in the following claims.

In the description herein, references to the description of "one embodiment," "an example," "a specific example" or the like are intended to mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above do not necessarily refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.

The preferred embodiments of the invention disclosed above are intended to be illustrative only. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise forms disclosed. Obviously, many modifications and variations are possible in light of the above teaching. The embodiments were chosen and described in order to best explain the principles of the invention and the practical application, to thereby enable others skilled in the art to best utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. The method for separating and culturing the human epidermal stem cells is characterized by comprising the following steps:

step five, preparing single cell suspension: observing cells in epidermal stem cell fluid by an inverted microscope, determining the amount of culture medium for resuspending cells after counting, adding appropriate amount of K-SFM culture solution, resuspending cells, counting, and adjusting cell concentrationDegree to 5X 10⁵Preparing single cell suspension per mL;

step six, incubation and culture at constant temperature: inoculating the single cell suspension into a six-well plate previously plated with type IV collagen, incubating overnight at constant temperature, changing the culture medium after 24 hours, and placing at 37 deg.C and 5% CO₂And (4) continuing culturing in the environment, changing the culture solution every 2-3 days, and carrying out passage when the cell growth is fused to 70%.

2. The method for isolated culture of human epidermal stem cells according to claim 1, wherein the specific process of step two is as follows: the epidermis after adding along feeder hopper (38) and the separation of corium layer starts first motor (21) in epidermis pruning chamber (20), first motor (21) are through first shaft coupling (22) drive pruning axle (23), pruning blade (24) rotate, a plurality of pruning blade (24) are pruned the epidermis and are cut, the air that blows out in sweeping case (27) makes the epidermis unordered float in epidermis pruning chamber (20), the epidermis after the pruning that reaches filter plate (25) filtration pore size requirement filters through filter plate (25) is in order falls into digester (42) through filtering passageway (32).

3. The method for isolated culture of human epidermal stem cells according to claim 1, wherein the specific process of step three is: 0.25 wt% of trypsin is added from a liquid inlet hopper (74), an electric motor (78) drives a cam (77) to rotate, the cam (77) pushes a push-pull head (76) in a reciprocating manner, the push-pull head (76) pushes a push-pull rod (75) to extrude the interior of a liquid injection cavity (72), and the trypsin is pushed into a liquid injection pipe (73) under pressure and uniformly drops into a digestion tank (42); the PTC heating piece (67) is electrified by a lead and then heats up, the PLC (62) is in communication connection with the temperature sensor (63), the PLC (62) is electrically connected with the PTC heating piece (67), when the temperature sensor (63) detects that the temperature reaches 37 ℃, a control signal is sent to the PLC (62), and the PLC (62) closes a conducting circuit of the PTC heating piece (67) so that the temperature of liquid in the digestion tank (42) is kept at 37 ℃;

after digestion is finished, the fetal calf serum is added from the liquid inlet hopper (74), the electric motor (78) drives the cam (77) to rotate, the cam (77) pushes the push-pull head (76) in a reciprocating manner, the push-pull head (76) pushes the push-pull rod (75) to extrude the interior of the liquid injection cavity (72), and the fetal calf serum is pushed into the liquid injection pipe (73) under pressure and uniformly dripped into the digestion tank (42); after the fetal calf serum is added, slowly pushing air through the liquid injection pipe (73) to blow and beat cells to obtain blowing and beating liquid; the blowing liquid is discharged into a centrifugal tube (57) through a one-way valve (47) and a liquid discharge tube (48).

4. The separation culture device for the human epidermal stem cells is characterized by comprising a separation culture box (10), wherein an epidermal trimming cavity (20), a blowing and filtering cavity (30), a digestion blowing and beating cavity (40) and a centrifugal separation cavity (50) are sequentially arranged in the separation culture box (10) from top to bottom; a first motor (21) is arranged above the epidermis trimming cavity (20), the first motor (21) is connected with a trimming shaft (23) extending into the epidermis trimming cavity (20) through a first coupler (22), a plurality of trimming blades (24) are arranged on the periphery of the trimming shaft (23), a filter plate (25) is arranged between the epidermis trimming cavity (20) and the purging filter cavity (30), a stabilizing plate (26) is arranged between the filter plate (25) and the inner wall of the separation culture box (10), and a purging box (27) facing the center of the epidermis trimming cavity (20) is arranged on the stabilizing plate (26); a plurality of partition plates (31) communicated with the filter plates (25) are arranged in the blowing filter cavity (30), a filter channel (32) is formed between the adjacent partition plates (31), and a feed hopper (38) is arranged at the top of the skin trimming cavity (20).

5. The isolated culture device of the human epidermal stem cells according to claim 4, wherein a plurality of blowers (33) are arranged in said purging filter chamber (30), a blast pipe (34) penetrating through said stabilizing plate (26) is arranged at the bottom of said purging box (27), said blast pipe (34) is connected to said blowers (33) via connecting flanges (35), a plurality of purging heads (36) are uniformly distributed on the upper surface of said purging box (27), and a plurality of purging holes (37) are distributed at the end of said purging heads (36).

6. The isolated culture device of human epidermal stem cells according to claim 4, wherein a digestion fixing frame (41) is provided at the center of the digestion blowing cavity (40), a constant temperature heating mechanism (60) is provided at both sides of the digestion fixing frame (41), a digestion tank (42) is fixed in the digestion fixing frame (41), and vent holes (43) are uniformly distributed on the wall of the digestion fixing frame (41); an enzyme injection mechanism (70) and a fetal calf serum injection mechanism (80) are respectively arranged at the two sides of the outer wall of the separation culture box (10) and at the digestion blow-beating cavity (40).

7. The isolated culture device of human epidermal stem cells according to claim 6, wherein the constant temperature heating mechanism (60) comprises a constant temperature heating box (61), a PLC (62) and a temperature sensor (63), the temperature sensor (63) is arranged on the outer wall of the digestion fixing frame (41), the constant temperature heating box (61) comprises a constant temperature heating frame (64) with openings at two sides, a plurality of aluminum pipes (65) are equidistantly distributed in the inner cavity of the constant temperature heating frame (64), a heat conduction pipe (66) is connected between each aluminum pipe (65) and the inner wall of the constant temperature heating frame (64) and between adjacent aluminum pipes (65), a PTC heating element (67) is inserted in each aluminum pipe (65), and a heat conduction hole (68) is formed in the wall of the constant temperature heating frame (64) in a penetrating manner.

8. The isolated culture device of human epidermal stem cells according to claim 7, wherein the bottom center of the digestion fixing frame (41) is connected with a second motor (45) through a second coupling (44), and a motor fixing frame (46) is arranged at the periphery of the second motor (45); the bottom of the digestion tank (42) is connected with a plurality of one-way valves (47) which penetrate through the digestion fixing frame (41), and the bottoms of the plurality of one-way valves (47) are connected with a liquid discharge pipe (48) which extends downwards.

9. The isolated culture apparatus of human epidermal stem cells according to claim 6, the structure of enzyme injection mechanism (70) and fetal calf serum injection mechanism (80) is the same, all include fixed plate (71), annotate liquid chamber (72) and annotate liquid pipe (73), fixed plate (71), annotate the outer wall connection of liquid chamber (72) and separation culture case (10), the one end and the notes liquid chamber (72) intercommunication of annotating liquid pipe (73), other end slope downwardly extending to the top of digestion tank (42), the top of annotating liquid chamber (72) is equipped with into liquid fill (74), it is equipped with push-and-pull rod (75) to keep away from separation culture case (10) outer wall adaptation in liquid chamber (72), the end connection of push-and-pull rod (75) has push-and-pull head (76) that stretches out to annotate liquid chamber (72), the outside of push-and-pull head (76) is equipped with cam (77), cam (77) are through arranging electric motor (78) drive rotation on fixed plate (71).

10. The human epidermal stem cell separation and culture device of claim 4, wherein a centrifugal separation mechanism is arranged in the centrifugal separation chamber (50), the centrifugal separation mechanism comprises a third motor (51), a vibration damping tank (52) and a centrifugal separation tank (53), the third motor (51) is arranged at the bottom of the separation culture tank (10), the vibration damping tank (52) and the centrifugal separation tank (53) are arranged at the bottom in the centrifugal separation chamber (50), the centrifugal separation tank (53) is arranged above the vibration damping tank (52), the third motor (51) is connected with a centrifugal shaft (55) extending into the vibration damping tank (52) through a third coupling (54), and a plurality of vibration damping springs (56) are arranged above the inner cavity of the vibration damping tank (52); the upper surface annular array of centrifugal separation case (53) distributes centrifuging tube (57) that a plurality of and fluid-discharge tube (48) correspond, and the inner chamber top of centrifuging tube (57) is equipped with screen cloth (58), and liquid distribution pipe (59) are connected with liquid collecting cavity (69) through liquid separating pipe (59) in the bottom of centrifuging tube (57).