CN107287153B - Differential digestion method of human epidermal cells - Google Patents
Differential digestion method of human epidermal cells Download PDFInfo
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- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
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- 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
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The application discloses a differential digestion method of human epidermal cells, and particularly relates to the field of histocytology. Human epidermal tissue from which subcutaneous loose connective tissue has been removed is digested with a D-H enzyme digest; adding serum stop solution after digestion is completed, and filtering and collecting the mixture to a centrifuge tube; pouring out the supernatant, gently blowing with PBS until the cell suspension is completely mixed, respectively taking appropriate amount of suspension in the upper half and the lower half, dripping different EP tubes, performing cell counting, and detecting cell viability; centrifuging, inoculating to epidermic cell culture solution, and concentrating with 5% CO at 37deg.C 2 Culturing in an incubator to obtain the epidermal cells. The method is simple and quick, has a large number of cells and high purity, and can establish a human epidermis cell bank with high activity for clinical transplantation and in vitro reconstruction of human skin models.
Description
Technical Field
The application relates to the field of histocytology, in particular to a differential digestion method of human epidermal cells. Background
Epidermal cells are epithelial cells of the skin surface layer, and mainly consist of keratinocytes and dendritic cells. Human epidermal cells play a vital role in wound healing, especially, the lack of epidermal keratinocytes of patients with severe burns has close relation with the duration of the patients, the occurrence of complications and the formation of post-scar, so that the application of tissue engineering technology to the separation, culture and preservation of epidermal cells in vitro is always a research hotspot of medical workers in clinic.
The current methods for epidermal cell isolation mainly include two main categories: tissue mass method and enzyme digestion method. The tissue block method is to directly separate the epidermal tissue after the skin tissue is disinfected and sterilized, cut the epidermal tissue into small blocks, culture the small blocks in a culture dish with gelatin spread downwards on the basal surface, culture the small blocks at 37 ℃, and digest and passage the small blocks with trypsin when the growth diameter of cells around the tissue blocks reaches 1-2 cm, so as to finally obtain the epidermal cells. The enzymatic method mainly uses enzymolysis to separate epidermis and dermis and obtain epidermis cells. In recent years, enzymatic hydrolysis has been continuously improved from the initial digestion with trypsin to the two-step enzymatic method using a combination of neutral protease and trypsin, which are widely used today, and the step digestion of the epidermis of the skin with a mixed enzyme of Dispase II and collagase I to obtain epidermal cells.
The application of patent application number 201410342022.6 reports a novel method for separating and culturing human epidermal cells in vitro, which comprises the following steps: collecting a normal skin tissue sample of a human body by about 0.5-5 cm < 2 >, and then placing the normal skin tissue sample into an F12 culture medium for cold preservation; weighing a certain amount of tissues, soaking in 200U/mL of PBS (poly (butylene succinate)) for 5 minutes, repeating the above operation once, cutting the tissues into homogenates, adding 1g of the homogenates into a centrifuge tube containing 20mL of digestive juice, and uniformly mixing; carrying out shaking digestion for 90-120 min in a water bath at 37 ℃; then adding trypsin with the final concentration of 0.05% to continue digestion for 30min, finally adding 100 mu L of 10mg/ml deoxyribonuclease to digest for 5min, adding the same volume of DMEM neutralizing digestive juice containing 10% FBS after tissue digestion, and repeatedly sucking and beating for 50-100 times; filtering the tissue suspension with a filter, and filtering the supernatant; washing the cell pellet once with DMEM, and centrifuging to discard the supernatant; the cell pellet was resuspended in epidermal cell culture medium and plated in a plating matrix-pretreated flask or dish at 37℃with 5% CO 2. The method saves time, has low cell purity, and has been cultured for 2 weeks to obtain 107 epidermis.
The patent application number is: 201510653684.6 reports a method for obtaining epidermal cells from autologous skin, which is: taking 0.1-2 cm < 2 > of skin of a person, soaking with 5mL of 1 XPBS for 15 minutes, repeating the above operation, washing, then digesting with a dispersing agent II, placing in a refrigerator at 4 ℃ for overnight, taking out the skin and dermis in the next day, tearing open the skin by forceps, shearing the skin to about 1mm < 3 >, washing with 1 XPBS, adding into a 15mL centrifuge tube, centrifuging at 1000rpm/min for 5 minutes, discarding the supernatant, adding 3mL of 0.25% (mass volume ratio) pancreatin for digestion for 1-60 minutes, wherein the pancreatin contains 0.01% EDTA in mass volume ratio; and (3) after digestion, sieving with a 40 mu m cell sieve, centrifuging, and re-suspending with PBS and normal saline to obtain 107-108 epidermal cells. Although the method has a large number of epidermal cells, the whole digestion time is long, and the method needs to be carried out overnight. Therefore, the establishment of the human epidermal cell separation method which is rapid, high in cell yield and high in cell purity has important practical significance.
Disclosure of Invention
The application provides a differential digestion method of human epidermal cells, which is simple and rapid, can obtain a large number of cells and has high purity, and can establish a human epidermal cell library with higher activity for clinical transplantation and in vitro reconstruction of human skin models.
In order to achieve the above purpose, the embodiment of the application adopts the following technical scheme:
the application provides a differential digestion method of human epidermal cells, which comprises the following steps:
(1) Preparing a sterile glass culture dish in an ultra clean bench, adding 10mL of 75% ethanol into one dish, adding 10mL of sterile PBS into each of the three dishes, immersing fresh human skin tissue into 75% ethanol solution for 30s, and spreading the curled portion of the dermis during the period to make the curled portion fully contact with ethanol to remove blood stains and reduce surface pollution;
(2) Transferring the sterilized tissue into PBS solution for pickling, transferring into another PBS dish for continuous cleaning until no blood stain exists after 1min, then spreading the surface of the tissue downwards in the dish, removing subcutaneous loose connective tissue, exposing compact connective tissue until the tissue block is not curled any more, transferring into fresh PBS for pickling after the process is completed;
(3) Cutting the tissue into a size of 0.2-2 cm < 2 >;
(4) Transferring the cut tissue blocks into 25mL serum bottles, adding 1mL of D-H enzyme digestive juice into each tissue block, completely immersing the tissue blocks by the digestive juice, sealing the bottle caps by using sealing films, placing the bottle caps into a refrigerator at 4 ℃, and shaking the serum bottles lightly to ensure that the tissues are uniformly dispersed at the bottom, and digesting for 8-10 hours, wherein the digestion process is in a dark place;
(5) Pouring the soaked tissue together with the D-H enzyme digestion solution into a sterile glass culture dish, tearing off the epidermis in brown color by forceps, collecting and putting the epidermis into a prepared 50mL primary digestion solution, transferring the primary digestion solution into a 37 ℃ carbon dioxide incubator, digesting for 10min, and vibrating once every 2 min;
(6) After digestion is completed, adding 10mL of serum stop solution into each 25mL of digestion bottle, blowing for 50 times, passing through a 200-mesh sieve, collecting filtrate into a 50mL centrifuge tube, adding the serum stop solution into the serum bottle, filtering and collecting the filtrate into the centrifuge tube, and centrifuging at 800rpm for 6min;
(7) Pouring out the supernatant after centrifugation, gently beating cells with PBS, sucking up and down (each time for 6 seconds), sucking down and beating up for 6-8 times (each time for 2 seconds) after sediment is blown off until the cell suspension is completely mixed, supplementing PBS according to the sediment amount, blowing up for 2-3 times (each time for 2 seconds) by using an elbow suction pipe, taking proper amounts of suspension from the upper half part and the lower half part of the cell suspension, respectively, dripping into different 1.5ml EP pipes, adding into a counting plate for cell counting, dyeing with trypan blue, detecting the activity of the cells, covering the centrifugal pipe, centrifuging at 800rpm, and centrifuging for 6 minutes;
(8) According to the counting result, inoculating according to 3.5E6/bottle, placing 10mL of epidermal cell culture solution in each bottle, and horizontally placing the bottle mouth of the culture bottle in a 5% CO2 incubator at 37 ℃ inwards for culturing;
(9) The obtained epidermal cells are subjected to Western blotting protein detection to obtain the expression level of the epidermal cell growth factor, and the gray value of the epidermal cells is displayed to reach over 9000 through a gray level analysis result.
Further, the D-H enzyme digestion solution consists of a Dispase enzyme and a Hyaluronidase enzyme, wherein the activity unit of the Dispase enzyme is 1U/mL, the mass fraction is 1%, and the concentration of the Hyaluronidase enzyme is 0.1-0.3 mg/mL.
Further, the primary digestive juice is pancreatin digestive juice, and 25mg of pancreatin is contained in each 100mL of pancreatin digestive juice.
Further, the serum stop solution is composed of 10% fetal bovine serum added to DMEM medium.
Further, the ratio of the enzyme activity to the area of the skin tissue was 5:1 (U/cm 2).
The application has the advantages that: the digestion method is simple and easy to operate, is easy to master, needs small tissue amount, can obtain the epidermal cells 109 with stronger activity from the skin tissue with the size of 0.2cm < 2 >, has short time consumption in the extraction process, does not have extra reagent, greatly reduces the events such as bacterial pollution caused by complex operation or exogenous protein, has small damage to the protein on the cell surface, can be used for subsequent cell surface molecular screening, cell drug sensitivity test, cell sorting and identification and construction of a human recombinant skin model, and has wide prospect in basic research and clinical application of the epidermal cells.
Drawings
FIG. 1 shows human epidermal cells isolated by the differential digestion method of human epidermal cells according to the embodiment of the present application.
Detailed Description
The technical solutions in the embodiments of the present application will be described in detail below with reference to the accompanying drawings in the embodiments of the present application.
Example 1
(1) Materials and methods
Tissue: normal adult foreskin tissue discarded in hospital operation
Epidermis culture solution: adding L-glutamine and bovine pituitary extract into KC culture medium;
(2) Cell isolation and culture
A. Preparing sterile glass culture dishes in an ultra-clean bench, adding 10mL of 75% alcohol into one dish, and adding 10mL sterile PBS solution into each of three dishes; taking the skin taking box out of the package, putting the skin taking box into an ultra-clean bench, taking out the elbow tweezers and the straight tweezers, and leaning against the culture dish containing alcohol after the fire;
B. spraying 75% alcohol on the bottle filled with tissues outside the super clean bench, wiping the junction between the bottle mouth and the bottle cap by using a gauze soaked with 75% alcohol, opening the bottle cap in the super clean bench, carefully taking out the foreskin tissues from the sterile forceps after the bottle mouth is over fire, immersing the foreskin tissues in 75% alcohol solution for 30s, and spreading the curled part of the dermis surface by using the turning part of the elbow forceps during the period to make the curled part fully contact with alcohol so as to remove blood stains and reduce surface pollution;
C. immersing the tissue in PBS solution until no blood stain exists, removing loose connective tissue in a plate by using elbow scissors until the tissue block is no longer curled, and cutting the tissue with a scalpel into fresh PBS with the size of about 3mm multiplied by 9mm after the tissue is completed;
D. transferring each 15 pieces of the cut tissue blocks into 1 25mL serum bottles, adding 15mLD-H enzyme digestion solution, completely immersing the tissue blocks in the digestion solution, sealing the tissue blocks with a sealing film, placing the tissue blocks in a refrigerator at 4 ℃ for 8 hours, and keeping away from light during the process of adding D-H enzyme digestion solution and digestion;
E. preparing 50mL sterile serum bottles in an ultra-clean workbench, adding primary digestive juice preheated at 37 ℃ with half of D-H enzyme digestive juice, taking out the serum bottles for soaking tissues from 4 ℃, pouring the tissue blocks and the D-H enzyme digestive juice into a sterile glass culture dish, making the surface brown, and carefully tearing off the surface by using sterile forceps;
F. collecting epidermis into prepared digestive juice, digesting for 10min in a carbon dioxide incubator at 37 ℃ with shaking every 2min, transferring to an ultra clean bench, adding 10mL serum stop solution into each 25mL digestive flask, and gently blowing with an elbow suction tube for 50 times (1 time every 2 seconds);
G. placing a sterile 200-mesh screen (74 μm) into a sterile glass culture dish, filtering, adding 5mL of serum stop solution into a serum bottle to collect residual cells on the bottle wall, and filtering and collecting; collecting filtrate into a 50mL centrifuge tube, adding 5mL serum stop solution into the culture dish to collect residual cells in the culture dish, transferring the liquid into the centrifuge tube, and centrifuging at 800rpm for 6min;
H. pouring out the supernatant after centrifugation, adding 5mL of PBS, taking an elbow suction pipe to gently blow cells, sucking up and down (each time for 6 seconds), sucking down and beating up for 6-8 times (each time for 2 seconds) until the cell suspension is completely mixed after sediment is blown off, supplementing PBS according to the sediment amount, blowing up for 2-3 times (each time for 2 seconds) by using the elbow suction pipe, taking proper amounts of suspension from the upper half part and the lower half part of the cell suspension respectively, dripping into different 1.5mLEP pipes, respectively taking 10 mu L of the suspension for carrying out technology, using 0.4% trypan blue for dyeing and detecting the cell viability, and using Western blotting protein for detecting the expression level of the epidermal cell growth factor;
I. according to the counting result, calculating the number of required T75 disposable culture flasks according to the cell inoculation density (3.5-3.75E6/flask), putting the T75 disposable culture flasks into an ultra-clean bench, and marking the cell batch at the bottom. And adding 8mL of culture solution into each T75 culture bottle, uniformly distributing the cell suspension into the culture bottles along the cell wall surface of the culture bottle, adding 10-mL of the culture solution into each bottle, vertically shaking the culture bottle for 3-5 times to uniformly mix all the culture solutions, and horizontally taking up the culture bottle and slightly shaking for 3-5 times to uniformly disperse the cells. The flask mouth was placed horizontally inward in a 37℃5% CO2 incubator for cultivation.
The human epidermal cells separated by the differential digestion method of human epidermal cells provided by the embodiment of the application are shown in figure 1, and the method is simple and rapid, the obtained cells are large in number and high in purity, and a human epidermal cell library with high activity can be established for clinical transplantation and in vitro reconstruction of human skin models.
The embodiment of the application provides a differential digestion method of human epidermal cells, which comprises the following steps: (1) Preparing a sterile glass culture dish in an ultra clean bench, adding 10mL of 75% ethanol into one dish, adding 10mL of sterile PBS into each of the three dishes, immersing fresh human skin tissue into 75% ethanol solution for 30s, and spreading the curled portion of the dermis during the period to make the curled portion fully contact with ethanol to remove blood stains and reduce surface pollution; (2) Transferring the sterilized tissue into PBS solution for pickling, transferring into another PBS dish for continuous cleaning until no blood stain exists after 1min, then spreading the surface of the tissue downwards in the dish, removing subcutaneous loose connective tissue, exposing compact connective tissue until the tissue block is not curled any more, transferring into fresh PBS for pickling after the process is completed; (3) Cutting the tissue into a size of 0.2-2 cm < 2 >; (4) Transferring the cut tissue blocks into 25mL serum bottles, adding 1mL of D-H enzyme digestive juice into each tissue block, completely immersing the tissue blocks by the digestive juice, sealing the bottle caps by using sealing films, placing the bottle caps into a refrigerator at 4 ℃, and shaking the serum bottles lightly to ensure that the tissues are uniformly dispersed at the bottom, and digesting for 8-10 hours, wherein the digestion process is in a dark place; (5) Pouring the soaked tissue together with the D-H enzyme digestion solution into a sterile glass culture dish, tearing off the epidermis in brown color by forceps, collecting and putting the epidermis into a prepared 50mL primary digestion solution, transferring the primary digestion solution into a 37 ℃ carbon dioxide incubator, digesting for 10min, and vibrating once every 2 min; (6) After digestion is completed, adding 10mL of serum stop solution into each 25mL of digestion bottle, blowing for 50 times, passing through a 200-mesh sieve, collecting filtrate into a 50mL centrifuge tube, adding the serum stop solution into the serum bottle, filtering and collecting the filtrate into the centrifuge tube, and centrifuging at 800rpm for 6min; (7) Pouring out the supernatant after centrifugation, gently beating cells with PBS, sucking up and down (each time for 6 seconds), sucking down and beating up for 6-8 times (each time for 2 seconds) after sediment is blown off until the cell suspension is completely mixed, supplementing PBS according to the sediment quantity, blowing up for 2-3 times (each time for 2 seconds) by using an elbow suction pipe, respectively taking a proper amount of suspension from the upper half part and the lower half part of the cell suspension, dripping into different 1.5mLEP pipes, adding into a counting plate for cell counting, dyeing with trypan blue, detecting the activity of the cells, covering the centrifugal pipe, centrifuging at 800rpm, and centrifuging for 6 minutes; (8) According to the counting result, inoculating according to 3.5E6/bottle, placing 10mL of epidermal cell culture solution in each bottle, and horizontally placing the bottle mouth of the culture bottle in a 5% CO2 incubator at 37 ℃ inwards for culturing; (9) The obtained epidermal cells are subjected to Western blotting protein detection to obtain the expression level of the epidermal cell growth factor, and the gray value of the epidermal cells is displayed to reach over 9000 through a gray level analysis result. Based on the description of the embodiment, the digestion method is simple and easy to operate, the required tissue quantity is small, the skin tissue with the size of 0.2cm < 2 > can obtain the epidermal cells 109 with stronger activity, the extraction process is short in time consumption, no extra reagent is needed, the events such as bacterial pollution caused by complex operation or exogenous proteins are greatly reduced, the damage to the protein on the cell surface is small, and the digestion method can be used for subsequent cell surface molecular screening, cell drug sensitivity test, cell sorting and identification and construction of a human body recombinant skin model and has wide prospects in basic research and clinical application of the epidermal cells.
The foregoing is merely illustrative of specific embodiments of the present application, and the scope of the present application is not limited thereto, but any changes or substitutions within the technical scope of the present application should be covered by the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.
Claims (4)
1. A method for differential digestion of human epidermal cells, comprising the steps of:
preparing a sterile glass culture dish in an ultra clean bench, adding 10mL of 75% ethanol into one dish, adding 10mL of sterile PBS into each of the three dishes, immersing fresh human skin tissue into 75% ethanol solution for 30s, and spreading the curled portion of the dermis during the period to make the curled portion fully contact with ethanol to remove blood stains and reduce surface pollution;
transferring the sterilized tissue into PBS solution for pickling, transferring into another PBS dish for continuous cleaning until no blood stain exists after 1min, then spreading the surface of the tissue downwards in the dish, removing subcutaneous loose connective tissue, exposing compact connective tissue until the tissue block is not curled any more, transferring into fresh PBS for pickling after the process is completed;
cutting the tissue into a size of 0.2-0.27 cm < 2 >;
transferring the cut tissue blocks into 25mL serum bottles, adding 1mL of D-H enzyme digestive juice into each tissue block, completely immersing the tissue blocks by the digestive juice, sealing the bottle caps by using sealing films, putting the bottle caps into a refrigerator at 4 ℃, and shaking the serum bottles lightly to ensure that the tissues are uniformly dispersed at the bottom, and digesting for 8-10 hours, wherein the digestion process is light-proof; wherein the D-H enzyme digestive juice consists of a Dispase enzyme and a Hyaluronidase enzyme, the activity unit of the Dispase enzyme is 1U/mL, the mass fraction is 1%, and the concentration of the Hyaluronidase enzyme is 0.1-0.3 mg/mL;
pouring the soaked tissue together with D-H enzyme digestive juice into a sterile glass culture dish, tearing off the epidermis with forceps, collecting and placing the epidermis into a digestion bottle containing prepared primary digestive juice, transferring the digestion bottle into a 37 ℃ carbon dioxide incubator, and digesting for 10min, wherein the primary digestive juice is oscillated once every 2min, and the quantity of the primary digestive juice is half of that of the D-H enzyme digestive juice;
after digestion is completed, adding 10mL of serum stop solution into each 25mL of digestion bottle, blowing for 50 times, passing through a 200-mesh sieve, collecting filtrate into a 50mL centrifuge tube, adding the serum stop solution into the serum bottles, filtering and collecting the filtrate into the centrifuge tube, and centrifuging at 800rpm for 6min;
pouring out the supernatant after centrifugation, gently beating cells with PBS, sucking up and down for each time for 6 seconds, sucking down and beating up for 6-8 times after sediment is blown off, each time for 2 seconds until the cell suspension is completely mixed, supplementing PBS according to the sediment quantity, blowing up for 2-3 times with an elbow suction pipe, each time for 2 seconds, taking proper amount of suspension from the upper half part and the lower half part of the cell suspension, dripping into different 1.5mLEP pipes, adding into a counting plate for cell counting, staining with trypan blue, detecting the activity of the cells, covering the centrifugal pipe, centrifuging at 800rpm, and centrifuging for 6 minutes;
according to the counting result, inoculating according to 3.5E6/bottle, placing 10mL of epidermal cell culture solution in each bottle, and horizontally placing the bottle mouth of the culture bottle in a 5% CO2 incubator at 37 ℃ inwards for culturing;
the obtained epidermal cells are subjected to Western blotting protein detection to obtain the expression level of the epidermal cell growth factor, and the gray value of the epidermal cells is displayed to reach over 9000 through a gray level analysis result.
2. The differential digestion method of human epidermal cells according to claim 1, wherein said primary digestive juice is pancreatin digestive juice comprising 25mg of pancreatin per 100mL of pancreatin digestive juice.
3. The method according to claim 1, wherein the serum stop solution is prepared by adding 10% fetal bovine serum to DMEM medium.
4. The method of differential digestion of human epidermal cells according to claim 1, wherein the ratio of the Dispase enzyme activity to the area of skin tissue is 5:1u/cm2.
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