CN111560346A - Method for efficiently extracting and proliferating autologous fibroblasts by explant adhesion method - Google Patents
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Abstract
The invention provides a method for efficiently extracting and proliferating autologous fibroblasts, which is to improve a scheme on a basic explant adhesion method so as to establish the acquisition and culture of primary fibroblasts more simply and efficiently. The invention has no damage to cells caused by a dissociation method to affect the activity, simultaneously improves the culture condition, overcomes the difficulty of obtaining the cells from the tissue block, improves the proliferation efficiency of low-density cells, and efficiently shortens the period of subcutaneous injection of autologous fibroblasts.
Description
Technical Field
The invention relates to a method for rapidly proliferating autologous fibroblasts in vitro by using an explant adhesion method, and belongs to the field of cell culture.
Background
Aging is not resistant, and no one can keep youth forever. With the increase of age, the viable and tender cells lose water, have small volume and are not full, the metabolism rate is gradually slowed down, and the cells gradually age naturally until apoptosis. On the other hand, the decline of stem cells in the body leads to the generation of new cells which can not completely replace old aged cells with aging and apoptosis in quantity, thus leading to the reduction of the number of effective cells of the body, the degeneration of elastic fibers and collagen fibers of the skin, the atrophy, the thinning and the pigmentation of the skin and the youth.
With the increase of social economy, the medical beauty industry is growing, and facial repair is the first place in the medical beauty industry, and more people choose to intervene in the aging of skin through medical means. At present, one of the means of the medical and American industry for treating facial aging is to substitute extracellular matrix components reduced by aged cells in the dermis of the skin by introducing biological agents such as collagen and hyaluronidase into the skin so as to achieve the effects of filling and removing wrinkles. The method is undeniably quick in effect and obvious in effect, but the components are also quickly degraded under the action of biological metabolism, and the treatment effect is not lasting. Meanwhile, the introduction of the non-self components easily causes the rejection of the organism and generates anaphylactic reaction.
The skin is composed of the epidermis and the dermis layers, and fibroblasts present in the dermis layers are an important secretory source of extracellular matrix. The amount of collagen secreted affects the thickness, elasticity, and luster of the skin. However, as the skin ages, fibroblasts in the dermis and collagen secreted by the fibroblasts are continuously reduced, so that the normal structure and physiological functions of the skin cannot be maintained. Therefore, the skin anti-wrinkle cream can supplement the number of skin fibroblasts, promote the secretion of collagen, and become a reliable means for removing wrinkles and treating depressed scars. While human skin tissue is readily available and has fewer ethical and repulsive problems than other sources, human fibroblasts isolated from skin tissue are a convenient material for future clinical use.
Disclosure of Invention
The invention provides a method for efficiently extracting and proliferating autologous fibroblasts by an explant adhesion method, which is characterized in that a tissue engineering technology is used for separating, purifying, culturing and amplifying skin tissues, and a fibroblast precursor cell injection is obtained and introduced into the dermis layer of the skin of a patient. The autologous fibroblasts do not have the rejection condition and can well grow in the dermis layer tissue of a patient, so that extracellular matrixes such as collagen, elastic fibers and the like are continuously secreted by the autologous aged fibroblasts, the thickness of the dermis is increased, and the elasticity and the glossiness of the skin are recovered. Or can be specifically introduced into the dermis of the damaged skin of the dented scar, and the fibroblasts can secrete extracellular matrix molecules and also generate complex cell growth factors, thereby having the efficacy of promoting wound healing and removing scars.
The explant adhesion method and the dissociation method are the most basic methods for obtaining fibroblasts from tissues at present, however, skin tissues are mostly taken from the parts of the back of the ear, the eyelid and the like of a patient, the size of the skin is limited, and the limited skin is easy to cause enzyme over-digestion in the extraction process of the tissue dissociation method, so that the activity of cells is reduced, and the growth state of the subsequent cells is influenced. The invention improves the scheme on the basis of an explant adhesion method, thereby establishing the acquisition and culture of primary fibroblasts more simply and efficiently.
In order to solve the above-mentioned purpose, the technical scheme adopted by the invention is as follows:
the method for extracting the fibroblasts has the innovation points that: the method comprises the following specific steps:
(1) skin and peripheral blood acquisition: collecting healthy people who have not taken aspirin and other antiplatelet and anticoagulant drugs within 2 weeks and have no systemic diseases, infectious diseases and venereal diseases, and 0.25cm behind ear or eyelid2～1cm240ml to 60ml of skin lumps and peripheral blood;
(2) platelet Rich Plasma (PRP) extraction: platelet plasma concentrate obtained by centrifuging peripheral blood; extracting peripheral blood with PRP at 22-24 deg.C for 6 hr, adding DMSO cryoprotectant with final concentration of 5-10 wt%, storing at-80 deg.C, and adding CaCl before use2Solution: adding 8-12wt.% CaCl into platelet plasma at a volume ratio of 1:92Adding the activated solution into a DMEM/F12 culture medium to enable the final concentration to be 1.0-3.0 × 1012plt/L; PRP is an autologous safe blood product, is rich in platelets, leukocytes, insulin-like growth factors (IGF) and the like, is a basic diffusant of fibroblasts, and is beneficial to the rapid proliferation of the fibroblasts;
(3) washing the leather blocks: rinsing the skin clot with normal saline, soaking in 75% ethanol for 30S for disinfection, rinsing the ethanol with normal saline, peeling off the blood vessel and connective tissue of the skin, and rinsing the rest skin tissue with normal saline for 7 times to thoroughly clean the skin tissue clot;
(4) coating and inoculating culture bottle: coating 5ug-10ug/ml fibrin solution at 2 deg.C-8 deg.C overnight, spreading the fibrin solution on the bottom of the culture bottle, and cutting the washed skin into pieces of 1mm with sterile scalpel3Inoculating small pieces of the above materials in a culture flask with tissue block distance of 5mm, and standing at 37 deg.C with 5% CO2The incubator was incubated for 2-4 hours to allow tissue to adhere to the bottom of the flask. The fibrin coating contributes to the adhesion of fibroblasts and the ability of the outer surface to expand;
(5) culturing by adding DMEM/F12 medium containing barley- β dextran into the culture flask, and culturing at 37 deg.C with 5% CO2Culturing in culture box, wherein the barley- β dextran has final concentration of 300-500 ug/ml, the time of day for the process from skin tissue block to cell acquisition is longer, while the barley- β dextran can promote the migration of human skin fibroblast, and shorten the time of day for cell migration from tissue;
(6) liquid changing, namely changing the same DMEM/F12 culture medium containing barley- β glucan every three days before the tissue blocks are not migrated out of the cells until adherent cells around the tissues are observed to grow, and changing the DMEM/F12 culture medium containing PRP with the final concentration of 1.0-3.0 × 10 PRP12plt/L. Standing at 37 deg.C and 5% CO2、2%-5%O2Culturing in an incubator. The tissue mass has a low number of cells in the early stage, which migrate out of the cells, a low number of cells, and a slow rate of expansion, in the case of a low cell density (<102Per cm2) Culturing in a hypoxic state can improve the proliferation rate of fibroblasts;
(7) passage: replacing the same DMEM/F12 culture medium containing PRP every 3 days, when the cell fusion degree reaches 80%, washing with normal saline for 2 times, adding trypsin solution with final concentration of 0.05wt.% for digestion for 2min, adding DMEM/F12 culture medium with the same volume for stopping digestion, collecting cells, which are P0 generation cells, and culturing at 10000/cm2The density of (2) is subjected to passage;
(8) and (3) identification: surface marker CD44 of extracted human fibroblast by flow detection method＋、CD29＋、Vimentin＋The expression level was measured.
The invention has the advantages that:
the invention utilizes fibrin to coat the culture bottle, improves the adhesion and the expansion force of fibroblasts, and simultaneously uses DMEM/F12 culture medium containing barley-beta glucan, improves the migration speed of the fibroblasts in the skin tissue block, greatly shortens the time for obtaining the cells from the tissue block, and particularly obviously improves the cell extraction speed of the aged skin tissue block from middle-aged and old people; meanwhile, the number of cells is limited in the early stage of obtaining the cells from a small amount of tissue blocks, the low-density culture influences the survival of the cells and greatly influences the proliferation efficiency, the method improves the cloning growth capacity of the cells under the low-density culture condition by reducing the oxygen concentration in the cell culture environment, improves the survival rate, stimulates the cells to rapidly expand by the blood plasma rich in platelets, and safely and efficiently obtains the fibroblasts with large number and high purity.
The method for extracting fibroblasts by adhering explants provided by the invention has the advantages that the damage of a dissociation method to the cells does not exist, the activity is not influenced, meanwhile, the improvement of the culture condition overcomes the difficulty of obtaining the cells from a tissue block, the low-density cell proliferation efficiency is improved, and the period of subcutaneous injection of autologous fibroblasts is efficiently shortened.
Drawings
FIG. 1 shows the cell migration ability of skin tissue under the combined action of fibrin and barley- β glucan, wherein A is an experimental group and B is a control group.
FIG. 2 is a bar graph of proliferation fold of low density fibroblasts after 7 days of culture with added PRP under hypoxic culture conditions versus no PRP under normoxic culture conditions.
FIG. 3 is a graph of the growth of low density fibroblasts after 7 days of culture with added PRP under hypoxic culture conditions versus no PRP under normoxic culture conditions.
FIG. 4 is a graph showing the results of flow-through assay of fibroblast cells P1 in example 1. A is isotype control group R1 target cell population, B is CD29+Isotype control, C is CD44+Isotype control, D is Vimentin+Isotype control.
FIG. 5 is a graph showing the results of flow-through assay of fibroblast cells P1 in example 1. E is the test group R1 target cell population, F is CD29+Positive forA population of cells, G being CD44+Positive cell population, H is Vimentin+A positive population of cells.
FIG. 6 is a graph showing the results of flow-through assay of fibroblast cells P1 in example 2. A is isotype control group R1 target cell population, B is CD29+Isotype control, C is CD44+Isotype control, D is Vimentin+Isotype control.
FIG. 7 is a graph showing the results of flow-through assay of fibroblast cells P1 in example 2. E is the test group R1 target cell population, F is CD29+Positive cell population, G is CD44+Positive cell population, H is Vimentin+A positive population of cells.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Example 1: comparing the advantages of the optimized explant adhesion method (experimental group) of the invention with the basic explant adhesion method (control group)
(1) Skin and peripheral blood acquisition: collecting 35 years old who has not taken aspirin and other related drugs with antiplatelet and anticoagulant effects within 2 weeks, and has no systemic diseases, infectious diseases and venereal diseases, and making into double eyelid operation person with eyelid of 0.25cm250ml of skin mass and peripheral blood;
(2) platelet Rich Plasma (PRP) extraction: 50ml of peripheral blood was transported to the laboratory under storage conditions at 22-24 ℃ and PRP extraction was completed within 2 hours. Centrifuging 50ml peripheral blood at 3000r/min for 3min, collecting all plasma layer, tunica albuginea layer and 2-3mm red blood cell layer, centrifuging for 2 times in another sterile blood collecting tube, centrifuging at 3000r/min for 10min, discarding 3/4 supernatant, and blowing and mixing the rest to obtain PRP. Adding a final concentration of 5wt.% DMSO cryoprotectant to the extracted PRP, and storing at-80 deg.C;
(3) coating a culture bottle: preparing 5ug/ml fibrin solution, sucking 2ml solution, spreading on the bottom of T25 culture bottle, and standing at 4 deg.C for coating overnight;
(4) washing the leather blocks: is measured at a distance of 0.25cm2Rinsing eyelid skin with normal saline, soaking in 75% ethanol for 30S for sterilization, rinsing with normal saline, and sterilizing with sterile handPeeling skin blood vessel and connective tissue with surgical instrument, rinsing the rest skin tissue with normal saline for 7 times, thoroughly cleaning skin tissue block, and cutting into 1mm pieces with disposable sterile surgical blade3Organizing the small blocks;
(5) inoculating and culturing: the experiment was divided into two groups, an experimental group and a control group, and was conducted. Experimental groups: the tissue blocks were inoculated at 5mm intervals into a fibrin solution-coated T25 flask and placed at 37 ℃ with 5% CO2The incubator was incubated for 2 hours to attach the tissue blocks to the bottom of the flask, 5mL of DMEM/F12 medium containing barley- β glucan was added to make the final concentration of barley- β glucan 400ug/mL, and as a control group, the tissue blocks were inoculated at a distance of 5mm into T25 flasks not coated with fibrin solution, and placed at 37 ℃ with 5% CO2The incubator is incubated for 2 hours to ensure that the tissue blocks are adhered to the bottom of the bottle, and 5ml of DMEM/F12 culture medium is added; simultaneously placing two groups of culture bottles at 37 ℃ with 5% CO2Culturing in an incubator, observing the condition that the tissue blocks migrate out of the cells every day, and replacing the corresponding culture medium every three days before the tissue blocks do not migrate out of the cells;
from the experimental results shown in fig. 1, the tissue blocks cultured to the 5d experimental group successfully migrated cells, and the control group showed no sign of cell migration, indicating that the experimental group utilized fibrin and barley-beta glucan to help improve the cell adhesion and migration ability;
(6) liquid changing: PRP is taken out from-80 ℃ in advance for redissolution according to CaCl2Solution: platelet plasma =1:9 volume ratio to PRP add 10wt.% CaCl as-prepared2Mixing the solutions, standing at room temperature for 10min, centrifuging at 12000r/min for 5min, extracting supernatant, and counting the supernatant by a hemograph to obtain PLT value for use, culturing the adherent cells of the experimental group and the control group in the step (5) until the cell fusion degree reaches 40-50%, digesting with trypsin of 0.05wt.% final concentration and re-suspending in DMEM/F12 medium, respectively, and simultaneously 9.6 × 10 for the experimental group and the control group from the corresponding cell suspension2Number of wells 1 plate 6 well cell culture plate was inoculated, and each well of the experimental group was changed to contain a final concentration of 1.5 × 1012plt/L PRP 2.5ml DMEM/F12 medium after standing at 37 ℃ 5% CO 22%O2Culturing in incubator, adding 2.5ml DMEM/F to each well of control group12 after the culture medium placed at 37 ℃ 5% CO 221%O2Culturing in an incubator. Changing the corresponding culture medium every three days and observing the growth condition of the cells in the two groups at the 7 th day (see figure 3), and respectively taking 1 culture hole in the two groups for counting and comparing the proliferation times of the cells in the two groups after 7 days of culture (see figure 2);
because the tissue of the experimental group in the step (5) migrates the cells earlier than the tissue of the control group and the number of the migrated cells is not consistent, the adherent cells in the experimental group and the control group in the step (5) are digested and collected after being cultured to a certain fusion degree and counted according to the time of 102/cm2The 6-well cell culture plates were inoculated in the same number, and the purpose was to compare the advantage of the experimental group and the control group in the same low-density cell number when the experimental group was cultured in the presence of hypoxia while adding PRP. FIG. 2 and FIG. 3 show that the experimental group can significantly improve the proliferation rate of fibroblasts under the action of low-oxygen environment culture in combination with PRP during low-density cell culture.
(7) Passage the remaining 5-well cells in the experimental group were replaced at a frequency of every 3d to a final concentration of 1.5 × 1012plt/L of PRP in DMEM/F12 medium, 5% CO at 37 ℃ 22%O2When the cells are cultured in an incubator until the cell fusion degree reaches 80 percent, the cells are harvested for P1 generation, and 2 × 10 is taken6The individual cells were used for flow detection with the result of CD44＋89.8%、CD29＋95.9%、Vimentin＋The flow results are shown in FIGS. 4-5 at 100%;
the results of fig. 4-5 show that the obtained fibroblast surface markers are all positive, indicating that the fibroblasts extracted by the present invention can meet the clinical application requirements.
Example 2
(1) Skin and peripheral blood acquisition: collecting 0.4cm behind ear of 30-year-old healthy female who has not taken aspirin and other related drugs with antiplatelet and anticoagulant effects within 2 weeks and has no systemic diseases, infectious diseases and venereal diseases250ml of skin mass and peripheral blood;
(2) platelet Rich Plasma (PRP) extraction: 50ml of peripheral blood was transported to the laboratory under storage conditions at 22-24 ℃ and PRP extraction was completed within 2 hours. Centrifuging 50ml of peripheral blood at 3000r/min for 3min, collecting all plasma layers, tunica albuginea layers and red blood cell layers within the range of 2-3mm to another sterile blood collection tube, centrifuging at 3000r/min for 10min, discarding 3/4 supernatant, blowing and uniformly mixing the rest to obtain PRP, adding 5% DMSO cryoprotectant into the extracted PRP, and storing at-80 deg.C for later use;
(3) coating a culture bottle: preparing 5ug/ml fibrin solution, sucking 2ml solution, spreading on the bottom of T25 culture bottle, and standing at 4 deg.C for coating overnight;
(4) washing the leather blocks: is measured at a distance of 0.4cm2Rinsing skin mass behind ear with normal saline, soaking in 75% ethanol for 30S for sterilization, rinsing with normal saline, peeling skin blood vessel and connective tissue with sterile surgical instrument, rinsing the rest skin tissue with normal saline for 7 times, thoroughly cleaning skin tissue mass, cutting into 1mm pieces with disposable sterile surgical blade3Organizing the small blocks;
(5) inoculating and culturing: the minced tissue pieces were inoculated at 5mm intervals into a fibrin solution-coated T25 flask and placed at 37 ℃ with 5% CO2The incubator was incubated for 2 hours to allow the tissue mass to adhere to the bottom of the flask, and then 5ml DMEM/F12 medium containing barley- β glucan at a final concentration of 400ug/ml was added to the flask and placed at 37 ℃ in 5% CO2Culturing in an incubator, and replacing the corresponding culture medium every three days at the earlier stage when the tissue blocks are not migrated out of the cells;
(6) liquid changing: until adherent cell growth around the tissue was observed, the PRP-containing DMEM/F12 medium was changed. PRP is taken out from-80 ℃ in advance for redissolution according to CaCl2Solution: platelet plasma =1:9 volume ratio to PRP add 10% CaCl as-prepared2Mixing the solutions, standing at room temperature for 10min, centrifuging at 12000r/min for 5min, collecting supernatant, counting by hematology instrument to obtain PLT value, and collecting PRP supernatant at 1.5 × 1012The final concentration of plt/L was added to 5ml DMEM/F12 medium in a T25 flask. Standing at 37 deg.C with 5% CO 22%O2Culturing in an incubator;
(7) passage: changing the same DMEM/F12 medium containing PRP in step (6) every 3 days, washing with normal saline for 2 times when the cell fusion degree reaches 80%, and addingDigesting with 0.05wt.% trypsin solution for 2min, adding equal volume of DMEM/F12 medium to stop digestion, collecting cells as P0 generation cells at 10000/cm2The density of the cells is subcultured in 1T 175 culture flask, the same DMEM/F12 culture medium containing PRP is replaced every 3 days, and when the cell fusion degree reaches 80%, cells of P1 generation are collected by trypsinization;
(8) identification, 2 × 10 is taken6The P1 fibroblasts collected by digestion are used for flow detection, and the flow detection result is CD44＋98.8%、CD29＋99.4%、Vimentin ＋100%, the flow results are shown in FIGS. 6-7;
the results of fig. 6-7 show that the obtained fibroblast surface markers are all positive, indicating that the fibroblasts extracted by the present invention can meet the clinical application requirements.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (1)
1. A method for efficiently extracting and proliferating autologous fibroblasts by an explant adhesion method is characterized by comprising the following steps:
(1) obtaining skin mass and peripheral blood: collecting healthy people without aspirin, other antiplatelet and anticoagulant effects within 2 weeks, and taking 0.25cm of eyelid or behind ear of healthy people without systemic diseases, infectious diseases and venereal diseases2～1cm240ml to 60ml of skin lumps and peripheral blood;
(2) platelet plasma extraction: platelet plasma obtained by centrifuging peripheral blood; extracting platelet plasma from peripheral blood at 22-24 deg.C within 6 hr, adding DMSO cryoprotectant with final concentration of 5-10wt.% and storing at-80 deg.C, and adding CaCl before use2Solution: platelet plasma volume ratio 1:9 to platelet plasma 8-12wt.% CaCl2Adding the activated solution into a DMEM/F12 culture medium to ensure that the final concentration of platelet plasma is 1.0-3.0 × 1012plt/L；
(3) Washing the leather blocks: rinsing the skin clot with normal saline, soaking in 75% ethanol for 30S for disinfection, rinsing the ethanol with normal saline, peeling off the blood vessel and connective tissue of the skin, and rinsing the rest skin tissue with normal saline for 7 times to thoroughly clean the skin tissue clot;
(4) coating and inoculating culture bottles: the culture bottle is coated with 5ug-10ug/ml fibrin solution at 2 deg.C-8 deg.C overnight, and the washed skin is cut into 1mm pieces with sterile scalpel3Inoculating small pieces of the above materials in a culture flask with tissue block distance of 5mm, and standing at 37 deg.C with 5% CO2Incubating the culture box for 2-4h to make the tissue adhere to the bottom of the bottle;
(5) culturing by adding DMEM/F12 medium containing barley β -dextran into the culture flask, and culturing at 37 deg.C with 5% CO2Culturing in incubator with barley β -dextran final concentration of 300-500 ug/ml;
(6) changing the culture medium, namely changing the same DMEM/F12 culture medium containing barley- β glucan every three days at the early stage until adherent cells around the tissues are observed to grow, and then changing the DMEM/F12 culture medium containing PRP (platelet-rich plasma) with the final concentration of 1.0-3.0 × 1012plt/L; standing at 37 deg.C and 5% CO2、2%-5%O2Culturing in an incubator;
(7) passage: replacing DMEM/F12 culture medium containing PRP every 3 days, when the cell fusion degree reaches 80%, washing with normal saline for 2 times, digesting with 0.05wt.% trypsin for 2min, adding the same volume of DMEM/F12 culture medium to stop digestion, collecting cells as P0 generation cells, and culturing at 10000/cm2The density of (2) is subjected to passage;
(8) and (3) identification: extracting human fibroblast surface marker CD44 by flow detection method＋、CD29＋、Vimentin＋The expression level was measured.
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