CN114686419B - Method and device for isolated culture of human epidermal stem cells - Google Patents
Method and device for isolated culture of human epidermal stem cells Download PDFInfo
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Abstract
The invention discloses a method and a device for separating and culturing 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, skin trimming and filtering, enzyme digestion and blowing, filtering and centrifugation, single cell suspension preparation and constant temperature incubation culture, and after the pediatric foreskin is collected, the method is cleaned and disinfected to remove subcutaneous tissues, and is different from the prior art in that the steps of skin trimming and filtering, enzyme digestion and blowing and filtering and centrifugation are completed through a separation culture device, the separation culture device can improve the skin trimming efficiency, keep the temperature in the enzyme digestion process constant, and automatically collect the skin stem cell liquid, so that the subsequent single cell suspension preparation and constant temperature incubation culture steps are facilitated.
Description
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, various tissue engineering skins are constructed according to the physiological structure of the skin, so that the problems of skin source shortage and prognosis scars are hopefully solved, and seed cells of the skin are mainly epidermal stem cells, fibroblasts and the like. Therefore, isolation and culture of epidermal stem cells are a necessary problem. In the prior report, the culture of human epidermal stem cells usually adopts neutral protease digestion overnight to separate the epidermal layer, and then uses 0.25% trypsin to digest for 10-15 min at 37 ℃ to separate the epidermal stem cells, but the defects of poor cell activity, low yield and the like are found in the practical application.
The method and the device for separating and culturing human epidermal stem cells in the prior art have the following technical problems: the efficiency of manually trimming the epidermis is low, and the temperature in the enzyme digestion process cannot be kept constant; the lack of an integrated device for trimming, enzyme digestion, filtering and centrifuging the epidermis reduces the efficiency of isolated culture.
Disclosure of Invention
The invention aims to provide a method and a device for separating and culturing human epidermal stem cells, which are used for solving the problems that in the prior art, the efficiency of manually trimming the epidermis is low and the temperature in the enzyme digestion process cannot be kept constant; the technical problem of reducing the efficiency of separation culture due to the lack of an integrated device for trimming, enzyme digestion, filtering and centrifuging the epidermis.
The aim of the invention can be achieved by the following technical scheme:
the method for separating and culturing the human epidermal stem cells comprises the following steps:
step one, selecting and preprocessing materials: selecting a prepuce of a child aged 2-6 years old, soaking the prepuce for 5-10 min by using iodophor, removing subcutaneous tissue under the aseptic condition, trimming the rest tissue to obtain a skin sheet with the size of 1cm multiplied by 1cm, digesting the skin sheet for 12-14 hours at 3-5 ℃ in a dark place by using Dispase enzyme, and separating to obtain epidermis and dermis;
step two, epidermis pruning and filtering: the epidermis is subjected to pruning and filtering by a separation culture device of human epidermis stem cells to obtain pruned epidermis;
step three, enzyme digestion and blowing: after trimming, the epidermis is digested by 0.25wt% trypsin for 10-15 min at 37 ℃; adding fetal calf serum to stop digestion, and blowing to obtain blowing liquid; wherein, the trypsin contains EDTA in an amount of 0.04 wt%;
step four, filtering and centrifuging: after filtering the blowing liquid, centrifugally separating for 5-8 min at 1400-1600 rpm, and standing and layering to obtain supernatant of an upper layer and epidermal stem cell liquid of a lower layer;
step five, single cell suspension preparation: observing cells in the epidermal stem cell liquid by an inverted microscope, counting, determining the culture medium amount of the resuspended cells, adding a proper amount of K-SFM culture solution, resuspending the cells, counting, and adjusting the cell concentration to 5×10 5 Preparing single cell suspension by using the single cell/mL;
step six, incubation and culture at constant temperature: inoculating single cell suspension into six-hole plate with IV type collagen plate, incubating at constant temperature overnight, changing culture solution after 24 hr, and placing at 37deg.C and 5% CO 2 And (3) continuing culturing in the environment, changing the culture solution every 2-3 days, and passaging when the cell growth is fused to 70%.
Further, the specific process of the second step is as follows: the epidermis after adding with the dermis layer separation along the feeder hopper, start the epidermis and prune the intracavity first motor, first motor passes through first shaft coupling drive 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 trim the intracavity unordered float of epidermis, reaches the pruning back epidermis of filter filtration pore size requirement and filters the back through the filter channel ordered and fall into the digester tank.
Further, the specific process of the third step is as follows: adding 0.25wt% 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 a push-pull rod by the push-pull head to squeeze towards the inside of a liquid injection cavity, pushing the trypsin into a liquid injection pipe under pressure, and uniformly dripping the trypsin into a digestion tank; the PTC heating element heats up after being electrified by a wire, 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 conduction 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 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 a push-pull rod by the push-pull head to squeeze the push-pull rod into a liquid injection cavity, pushing the fetal calf serum into a liquid injection pipe under pressure, and uniformly dripping the fetal calf serum into a digestion tank; after the fetal calf serum is added, slowly pushing air into a liquid injection pipe to blow cells to obtain a blowing liquid; the blowing liquid is discharged into the centrifuge tube through the one-way valve and the liquid discharge pipe.
The separation culture device for the human epidermal stem cells comprises a separation culture box, wherein an epidermal trimming cavity, a purging filter cavity, a digestion and 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 coupling, 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 a purging filter cavity, a stabilizing plate is arranged between the filter plate and the inner wall of a separation incubator, and a purging box facing the center of the epidermis trimming cavity is arranged on the stabilizing plate; a plurality of division plates communicated with the filter plates are arranged in the purging filter cavity, a filter channel is formed between every two adjacent division plates, and a feed hopper is arranged at the top of the epidermis trimming cavity.
Further, be equipped with a plurality of air-blower in the blowing filter chamber, the bottom of blowing the case is equipped with the blast pipe that runs through the firm board, and the blast pipe passes through flange and air-blower intercommunication, and the upper surface evenly distributed who blows the case has a plurality of blowing heads, and the tip of blowing head distributes a plurality of blowing holes.
Further, 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 ventilation holes are uniformly distributed in the wall part of the digestion fixing frame; the two sides of the outer wall of the separation incubator, which are positioned in the digestion blowing cavity, are respectively provided with an enzyme injection mechanism and a fetal bovine serum injection mechanism.
Further, constant temperature heating mechanism includes constant temperature heating cabinet, PLC controller and temperature sensor, and temperature sensor locates the outer wall of digestion fixed frame, and constant temperature heating cabinet includes both sides open-ended constant temperature heating frame, and constant temperature heating frame inner chamber equidistance distributes a plurality of aluminum pipes, is connected with the heat pipe between the inner wall of aluminum pipe and constant temperature heating frame, between the adjacent aluminum pipe, has inserted 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 bottom center of the digestion fixing frame is connected with a second motor through a second coupler, and the periphery of the second motor is provided with a motor fixing frame; the bottom of the digestion tank is connected with a plurality of check valves penetrating through the digestion fixing frame, and the bottoms of the check valves are connected with a liquid discharge pipe extending downwards.
Further, the structure of enzyme injection mechanism and child calf serum injection mechanism 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 separation incubator, annotate the one end and annotate the liquid chamber intercommunication of liquid pipe, the other end slope downwardly extending to the top of digester, annotate the top in liquid chamber and be equipped with the feed liquor bucket, annotate the liquid intracavity and keep away from the adaptation of separation incubator outer wall and be equipped with the push-and-pull rod, the end connection of push-and-pull rod has the push-and-pull head that stretches out the notes 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.
Further, a centrifugal separation mechanism is arranged in the centrifugal separation cavity, the centrifugal separation mechanism comprises a third motor, a vibration damping box and a centrifugal separation box, the third motor is arranged at the bottom of the separation incubator, 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 liquid collecting chamber through the fluid-discharge tube.
The invention has the following beneficial effects:
1. the separating and culturing method for human epidermal stem cells comprises the steps of collecting pediatric foreskin, cleaning, sterilizing and removing subcutaneous tissues, and completing the steps of epidermal trimming and filtering, enzyme digestion and blowing and filtering and centrifuging by a separating and culturing device, wherein the separating and culturing device can improve the epidermal trimming efficiency, keep the temperature constant in the enzyme digestion process, automatically collect the epidermal stem cell liquid, facilitate the subsequent single cell suspension preparation and constant temperature incubation culturing process.
2. The utility model provides a separation culture apparatus of human epidermis stem cell, the first motor of cuticle pruning intracavity starts the back, along the feeder hopper add with the epidermis after the dermis layer separation, first motor passes through first shaft coupling drive pruning axle, prune the blade and rotate, a plurality of prune the blade and prune the cutting to the epidermis, the air that blows out from blowing the incasement makes the epidermis trim the unordered float in the intracavity of epidermis, increases the contact probability of pruning blade and epidermis, improves the pruning efficiency of epidermis. The structure setting in the epidermis pruning chamber makes the epidermis after with the dermis layer separation, prunes efficiently, and the post-pruning size is even.
3. After current is introduced into the PTC heating elements, the PTC heating elements heat up to generate heat, the heat is conducted through the aluminum pipe and the heat conducting pipe and then is diffused through the heat conducting holes, so that the temperature in the digestion blowing cavity rises, the digestion tank absorbs heat to heat up to enable the epidermis and enzyme liquid to rise to 37 ℃, and the intelligent control of the PLC and the temperature sensor is combined, so that the liquid temperature in the digestion tank is kept at 37 ℃, and the stability of the enzyme digestion process is kept.
Drawings
In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a flow chart of a method for isolated culture of human epidermal stem cells in an embodiment of the present invention;
FIG. 2 is a schematic diagram showing the structure of a culture apparatus for isolated culture of human epidermal stem cells according to 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 diagram of an assembled structure of a purge bin and a blower tube, and a connecting flange in an embodiment of the present invention;
fig. 5 is a schematic diagram of an assembly structure of a digestion fixing frame, a digestion tank and a second motor in the embodiment of the invention;
FIG. 6 is a schematic view of a constant temperature heating box according to an embodiment of the present invention;
FIG. 7 is a schematic 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 invention;
FIG. 9 is a schematic diagram showing the structure of an enzyme injection mechanism or a fetal bovine serum injection mechanism in an embodiment of the present invention.
In the figure: 10. separating the incubator; 20. a epidermis clipping 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. a purge tank; 30. purging the filter chamber; 31. a partition plate; 32. a filtration channel; 33. a blower; 34. a blast pipe; 35. a connecting flange; 36. a purge head; 37. a purge hole; 38. a feed hopper; 40. digesting and blowing the cavity; 41. digesting the fixed 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 mandrel is detached; 56. a vibration damping spring; 57. centrifuging tube; 58. a screen; 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 pipe; 66. a heat conduction pipe; 67. a PTC heating element; 68. a heat transfer hole; 69. a liquid collection cavity; 70. an enzyme injection mechanism; 71. a fixing plate; 72. a liquid injection cavity; 73. a liquid injection pipe; 74. a liquid inlet hopper; 75. a push-pull rod; 76. a push-pull head; 77. a cam; 78. an electric motor; 80. a fetal bovine serum injection mechanism.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
As shown in fig. 1, the embodiment provides a method for isolated culture of human epidermal stem cells, comprising the following steps:
step one, selecting and preprocessing materials: selecting a prepuce of a child aged 2-6 years old, soaking the prepuce for 5-10 min by using iodophor, removing subcutaneous tissue under the aseptic condition, trimming the rest tissue to obtain a skin sheet with the size of 1cm multiplied by 1cm, digesting the skin sheet for 12-14 hours at 3-5 ℃ in a dark place by using Dispase enzyme, and separating to obtain epidermis and dermis;
step two, epidermis pruning and filtering: the epidermis is subjected to pruning and filtering by a separation culture device of human epidermis stem cells to obtain pruned epidermis;
step three, enzyme digestion and blowing: after trimming, the epidermis is digested by 0.25wt% trypsin for 10-15 min at 37 ℃; adding fetal calf serum to stop digestion, and blowing to obtain blowing liquid; wherein, the trypsin contains EDTA in an amount of 0.04 wt%.
Step four, filtering and centrifuging: after filtering the blowing liquid, centrifugally separating for 5-8 min at 1400-1600 rpm, and standing and layering to obtain supernatant of an upper layer and epidermal stem cell liquid of a lower layer;
step five, single cell suspension preparation: observing cells in the epidermal stem cell liquid by an inverted microscope, counting, determining the culture medium amount of the resuspended cells, adding a proper amount of K-SFM culture solution, resuspending the cells, counting, and adjusting the cell concentration to 5×10 5 Preparing single cell suspension by using the single cell/mL;
step six, incubation and culture at constant temperature: inoculating single cell suspension into six-hole plate with IV type collagen plate, incubating at constant temperature overnight, changing culture solution after 24 hr, and placing at 37deg.C and 5% CO 2 And (3) continuing culturing in the environment, changing the culture solution every 2-3 days, and passaging when the cell growth is fused to 70%.
During subculture, 0.25% trypsin is used to digest for about 5min, digestion is stopped, adherent cells are gently blown with a suction tube, cell suspension is collected, and centrifugation is carried out at 1000rpm for 5min, and the cells are discardedCells were cleared, resuspended and counted. According to the following steps: 3 ratio of passaging at 37deg.C, 5% CO 2 Culturing in a constant temperature incubator. And (5) observing the cell morphology and growth condition under an inverted microscope at regular intervals, and changing the liquid according to the growth condition.
The separation culture method of the human epidermal stem cells comprises the steps of material selection, pretreatment, epidermal trimming and filtering, enzyme digestion and blowing, filtering and centrifugation, single cell suspension preparation and constant temperature incubation culture, and after the pediatric foreskin is collected, the method is different from the prior art in that the steps of epidermal trimming and filtering, enzyme digestion and blowing and filtering and centrifugation are completed through a separation culture device, the separation culture device can improve the epidermal trimming efficiency, keep the temperature in the enzyme digestion process constant, and automatically collect the epidermal stem cell liquid, so that the subsequent single cell suspension preparation and constant temperature incubation culture process are facilitated.
Referring to fig. 2-9, the specific process of the second step is: the epidermis separated from the dermis layer is added along the feeding 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 coupling 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 in the epidermis trimming cavity 20 in an unordered manner, and the trimmed epidermis meeting the filter hole size requirement of the filter plate 25 is filtered by the filter plate 25 and then sequentially falls into the digestion tank 42 through the filter channel 32.
The specific process of the third step is as follows: 0.25wt% 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 squeeze towards the inside of 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 element 67 heats up after being electrified by a wire, the PLC 62 is in communication connection with the temperature sensor 63, the PLC 62 is electrically connected with the PTC heating element 67, when the temperature sensor 63 detects that the temperature reaches 37 ℃, a control signal is sent to the PLC 62, the PLC 62 closes a conduction circuit of the PTC heating element 67, so that the temperature of liquid in the digestion tank 42 is kept at 37 ℃;
after digestion is finished, adding fetal calf serum from a liquid inlet hopper 74, driving a cam 77 to rotate by an electric motor 78, pushing a push-pull head 76 by the cam 77 in a reciprocating manner, pushing a push-pull rod 75 by the push-pull head 76 to squeeze towards the inside of a liquid injection cavity 72, pushing the fetal calf serum into a liquid injection pipe 73 under pressure, and uniformly dripping into a digestion tank 42; after the fetal calf serum is added, slowly pushing air into the cell blowing device through the liquid injection pipe 73 to obtain blowing liquid; the blowing liquid is discharged into the centrifuge tube 57 through the check valve 47 and the liquid discharge pipe 48.
The specific process of the fourth step is as follows: the plurality of centrifuge tubes 57 filter and remove impurities from the blow-off liquid flowing in from the liquid discharge pipe 48 through the screen 58, the third motor 51 drives the centrifugal shaft 55 to rotate through the third coupler 54, the centrifugal shaft 55 drives the vibration damping box 52 and the centrifugal separation box 53 to rotate, the blow-off liquid in the centrifuge tubes 57 is centrifugally separated, supernatant of an upper layer and epidermal stem cell liquid of a lower layer are obtained through standing and layering, and the epidermal stem cell liquid enters the liquid collecting cavity 69 through the liquid separating pipe 59.
Example 2
As shown in fig. 2-3, the embodiment provides a separation culture device for human epidermal stem cells, which is used for completing the steps of epidermal trimming and filtering, enzyme digestion and blowing and filtering and centrifugation in the process of separating and culturing human epidermal stem cells, and comprises a separation culture box 10, wherein an epidermal trimming cavity 20, a purging filter cavity 30, a digestion and blowing cavity 40 and a centrifugation cavity 50 are sequentially arranged in the separation culture box 10 from top to bottom. The top of cuticle trimming chamber 20 is equipped with first motor 21, and first motor 21 is connected with the trimming axle 23 that stretches into cuticle trimming chamber 20 through first shaft coupling 22, and the periphery of trimming axle 23 is equipped with a plurality of trimming blades 24, is equipped with filter 25 between cuticle trimming chamber 20 and the purge filter chamber 30, is equipped with firm board 26 between filter 25 and the inner wall of separation incubator 10, is equipped with the purge case 27 towards cuticle trimming chamber 20 center on the firm board 26. A plurality of partition plates 31 communicated with the filter plates 25 are arranged in the purging filter chamber 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 epidermis trimming chamber 20. Wherein, the size of the filter holes of the filter plate 25 is 1-2 mm, the stabilizing plate 26 is obliquely downwards arranged from the side close to the inner wall of the separation incubator 10 to the side far from the inner wall of the separation incubator 10, and the interval between the adjacent partition plates 31 is 3-5 mm.
In the separating and culturing device for human epidermal stem cells in this embodiment, after the first motor 21 in the epidermal trimming cavity 20 is started, the epidermis separated from the dermis layer is added along the feeding hopper 38, the first motor 21 drives the trimming shaft 23 and the trimming blades 24 to rotate through the first coupling 22, the plurality of trimming blades 24 trim and cut the epidermis, air blown out from the blowing box 27 enables the epidermis to float randomly in the epidermal trimming cavity 20, the contact probability between the trimming blades 24 and the epidermis is increased, the trimming efficiency of the epidermis is improved, and after trimming, the epidermis which meets the size requirement of the filter holes of the filter plate 25 is filtered by the filter plate 25, and then falls into the blowing and filtering cavity 30 orderly through the filter channel 32. The structural arrangement in the epidermis trimming chamber 20 enables the epidermis to be separated from the dermis layer with high trimming efficiency and uniform size after trimming.
As shown in fig. 3-4, a plurality of blowers 33 are arranged in the purging filter cavity 30, a blower pipe 34 penetrating through the stabilizing plate 26 is arranged at the bottom of the purging box 27, the blower 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 parts of the purging heads 36. After the blower 33 blows air into the purge box 27 through the blower pipe 34, the air blows dense air in different directions through purge holes 37 on the purge heads 36, so that the epidermis floats in disorder in the epidermis trimming cavity 20, and the contact area with the trimming blade 24 is increased.
As shown in fig. 2 and fig. 5-6, a digestion fixing frame 41 is arranged in the center of the digestion blowing chamber 40, constant-temperature heating mechanisms 60 are arranged on two sides of the digestion fixing frame 41, a digestion tank 42 is fixed in the digestion fixing frame 41, and ventilation holes 43 are uniformly distributed in the wall of the digestion fixing frame 41. The outer wall of the separation incubator 10 is provided with an enzyme injection mechanism 70 and a fetal bovine serum injection mechanism 80 on both sides thereof located in the digestion and blowing chamber 40, respectively.
A digestion tank 42 fixed in the digestion fixing frame 41 is convenient for receiving falling trimmed epidermis and digestion after enzyme addition and blowing after fetal calf serum addition. The enzyme injection mechanism 70 can add an enzyme to the digestion tank 42, and the fetal bovine serum injection mechanism 80 can add a fetal bovine serum to the digestion tank 42.
The constant temperature heating mechanism 60 comprises a constant temperature heating box 61, a PLC controller 62 and a temperature sensor 63, wherein the temperature sensor 63 is arranged on the outer wall of the digestion fixing frame 41, the constant temperature heating box 61 comprises constant temperature heating frames 64 with openings on two sides, a plurality of aluminum pipes 65 are distributed in the inner cavity of each constant temperature heating frame 64 at equal intervals, heat conducting pipes 66 are connected between the aluminum pipes 65 and the inner wall of each constant temperature heating frame 64 and between the adjacent aluminum pipes 65, PTC heating pieces 67 are inserted in the aluminum pipes 65, and heat transfer holes 68 are formed in the wall portions of the constant temperature heating frames 64 in a penetrating mode.
The PTC heating element 67 heats up after being electrified by a wire, the PLC controller 62 is in communication connection with the temperature sensor 63, the PLC controller 62 is electrically connected with the PTC heating element 67, when the temperature sensor 63 detects that the temperature reaches 37 ℃, a control signal is sent to the PLC controller 62, the PLC controller 62 closes a conduction circuit of the PTC heating element 67, and the intelligent control enables the liquid temperature in the digestion tank 42 to be kept at 37 ℃.
When electric current is introduced into the plurality of PTC heating elements 67, the PTC heating elements 67 heat up to generate heat, the heat is conducted through the aluminum tubes 65 and the heat conducting tubes 66 and then is diffused through the heat conducting holes 68, so that the temperature in the digestion and blowing cavity 40 rises, and the digestion tank 42 absorbs heat and rises to heat the epidermis and the enzyme liquid to 37 ℃. The intelligent control of the PLC 62 and the temperature sensor 63 is combined, so that the temperature of the liquid in the digestion tank 42 is kept at 37 ℃ and the stability of 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 provided at the periphery of the second motor 45. The bottom of the digestion tank 42 is connected with a plurality of check valves 47 penetrating the digestion fixing frame 41, and the bottom of the check valves 47 is connected with a drain 48 extending downward. The second motor 45 drives the digestion fixed frame 41 to rotate through the second coupler 44, and the digestion fixed frame 41 drives the digestion tank 42 to rotate, so that digestion blowing efficiency is improved; the digested and blown liquid 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 bovine serum injection mechanism 80 have the same structure and comprise a fixed plate 71, a liquid injection cavity 72 and a liquid injection pipe 73, wherein the fixed plate 71, the liquid injection cavity 72 and the outer wall of the separation incubator 10 are connected, one end of the liquid injection pipe 73 is communicated with the liquid injection cavity 72, the other end of the liquid injection pipe obliquely extends downwards to the upper part of the digestion tank 42, a liquid inlet hopper 74 is arranged at the top of the liquid injection cavity 72, a push-pull rod 75 is arranged in the liquid injection cavity 72 far away from the outer wall of the separation incubator 10 in an adapting manner, the end part of the push-pull rod 75 is connected with a push-pull head 76 extending out of the liquid injection cavity 72, a cam 77 is arranged at the outer side of the push-pull head 76, and the cam 77 is driven to rotate by an electric motor 78 arranged on the fixed plate 71.
In the process of driving the cam 77 to rotate by the electric motor 78, the cam 77 reciprocally pushes the push-pull head 76, the push-pull head 76 pushes the push-pull rod 75 to squeeze into the liquid injection cavity 72, and after enzyme or fetal bovine serum is added from the liquid inlet hopper 74, the enzyme or fetal bovine 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 cavity 50, the centrifugal separation mechanism comprises 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 cavity 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. The upper surface annular array of centrifugal separation case 53 distributes a plurality of centrifuging tubes 57 that correspond with the fluid-discharge tube 48, 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 chamber 69 through separating tube 59. Wherein the mesh size of the screen 58 is 200 to 300 mesh.
The third motor 51 drives the centrifugal shaft 55 to rotate through the third coupling 54, the centrifugal shaft 55 drives the vibration damping box 52 and the centrifugal separation box 53 to rotate, the vibration damping springs 56 can relieve vibration generated by the centrifugal separation box 53 in the rotation process, the plurality of centrifugal tubes 57 filter and remove impurities from the blowing liquid flowing in from the liquid discharge pipe 48 through the screen 58, centrifugal separation is carried out, supernatant liquid of an upper layer and epidermal stem cell liquid of a lower layer are obtained after layering, and the epidermal stem cell liquid enters the liquid collecting cavity 69 through the liquid separating pipe 59 for the next treatment process.
Experimental example
Immunocytochemistry staining is carried out on the epidermal stem cells isolated and cultured in the embodiment 1, cell patches are washed by PBS, 4% paraformaldehyde is fixed for 20min, immunocytochemistry staining detection of beta 1 integrin and CKl is carried out by adopting an SP method according to the using instruction steps of a reagent, DAB color development is carried out after primary antibody and secondary antibody are added dropwise, counterstaining, dehydration, transparency and sealing are carried out, and observation is carried out under a mirror. The epidermal stem cell markers beta 1 integrin and CKl9 all react positively.
The foregoing is merely illustrative of the structures of this invention and various modifications, additions and substitutions for those skilled in the art can be made to the described embodiments without departing from the scope of the invention or from the scope of the invention as defined in the accompanying claims.
In the description of the present specification, the descriptions of the terms "one embodiment," "example," "specific example," and the like, 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 present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. 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 only to assist in the explanation of the invention. The preferred embodiments are not intended to be exhaustive or to limit the invention to the precise form 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 understand and utilize the invention. The invention is limited only by the claims and the full scope and equivalents thereof.
Claims (1)
1. 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 purging filter cavity (30), a digestion blowing 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 coupling (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 a purging filter cavity (30), a stabilizing plate (26) is arranged between the filter plate (25) and the inner wall of the separation incubator (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 purging 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 epidermis trimming cavity (20);
a plurality of blowers (33) are arranged in the purging filter cavity (30), a blast pipe (34) penetrating through the stabilizing plate (26) is arranged 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 parts of the purging heads (36);
a digestion fixing frame (41) is arranged in the center of the digestion blowing cavity (40), constant-temperature heating mechanisms (60) are arranged on two sides of the digestion fixing frame (41), a digestion tank (42) is fixed in the digestion fixing frame (41), and ventilation holes (43) are uniformly distributed in the wall part of the digestion fixing frame (41); the two sides of the outer wall of the separation incubator (10) are respectively provided with an enzyme injection mechanism (70) and a fetal bovine serum injection mechanism (80) which are positioned in the digestion blowing cavity (40);
the constant temperature heating mechanism (60) comprises a constant temperature heating box (61), a PLC (programmable logic controller) (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 constant temperature heating frames (64) with openings at two sides, a plurality of aluminum pipes (65) are distributed in the inner cavity of each constant temperature heating frame (64) at equal intervals, heat conduction pipes (66) are connected between each aluminum pipe (65) and the inner wall of each constant temperature heating frame (64) and between every two adjacent aluminum pipes (65), PTC heating pieces (67) are inserted in each aluminum pipe (65), and heat transfer holes (68) are formed in the wall portion of each constant temperature heating frame (64) in a penetrating mode;
the bottom center of the digestion fixing frame (41) is connected with a second motor (45) through a second coupler (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) penetrating through the digestion fixing frame (41), and the bottoms of the one-way valves (47) are connected with a liquid discharge pipe (48) extending downwards;
the enzyme injection mechanism (70) and the fetal bovine serum injection mechanism (80) are identical in structure and comprise a fixed plate (71), a liquid injection cavity (72) and a liquid injection pipe (73), the fixed plate (71), the liquid injection cavity (72) and the outer wall of the separation incubator (10) are connected, one end of the liquid injection pipe (73) is communicated with the liquid injection cavity (72), the other end of the liquid injection pipe obliquely extends downwards to the upper part of the digestion tank (42), a liquid inlet hopper (74) is arranged at the top of the liquid injection cavity (72), a push-pull rod (75) is arranged in the liquid injection cavity (72) far away from the outer wall of the separation incubator (10) in an adapting mode, the end portion of the push-pull rod (75) is connected with a push-pull head (76) extending out of the liquid injection cavity (72), a cam (77) is arranged on the outer side of the push-pull head (76), and the cam (77) is driven to rotate by an electric motor (78) arranged on the fixed plate (71);
the centrifugal separation device is characterized in that a centrifugal separation mechanism is arranged in the centrifugal separation cavity (50), the centrifugal separation mechanism comprises 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 inner bottom of the centrifugal separation cavity (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); the upper surface annular array of centrifugal separation case (53) distributes a plurality of centrifuging tube (57) that correspond with fluid-discharge tube (48), 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 fluid-separation tube (59).
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