CN109771697B - Dermal fibroblast skin sheet and construction method and application thereof - Google Patents
Dermal fibroblast skin sheet and construction method and application thereof Download PDFInfo
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- CN109771697B CN109771697B CN201811635902.7A CN201811635902A CN109771697B CN 109771697 B CN109771697 B CN 109771697B CN 201811635902 A CN201811635902 A CN 201811635902A CN 109771697 B CN109771697 B CN 109771697B
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Abstract
The invention provides a method for constructing an autologous dermal fibroblast skin sheet, which comprises the following steps: inoculating the dermal fibroblast expanded in vitro to the amniotic tissue which is subjected to decellularization, degreasing and nucleic acid removal treatment for cell solidification, constructing a composite structure of dermal fibroblast and amniotic tissue, and enriching high-purity cells under the cell culture condition to obtain the autologous dermal fibroblast skin sheet. The invention also provides an autologous dermal fibroblast skin sheet constructed by the method. The autologous dermal fibroblast skin sheet can be used for burn wounds, large-area skin grafting and other clinical requirements for urgently needing large-area skin defects, has the advantages of low rejection, sustainable supply and the like, and has wide application prospect.
Description
Technical Field
The invention relates to the technical field of in-vitro production of dermal fibroblasts, in particular to a dermal fibroblast skin sheet and a construction method and application thereof.
Background
The in vitro production technology of the autologous dermal fibroblasts: the technology for the in vitro production of autologous cells is now well established. The fibroblast in-vitro proliferation technology of adult autologous dermis already meets the industrialization requirement, the injection of the product is already on the market abroad, and similar products appear in China. At present, the large-scale production of adult dermal fibroblasts develops towards the direction of high efficiency, high purity and high activity; the product can be directly used for preparing injections, and can also be used as a main raw material component of other preparations.
The technique for constructing the solid cells by the de-epithelialized amnion comprises the following steps: the cell solidification technology is developed to date and gradually divided into two types, one is cell removal and cell addition treatment by using a biological tissue membrane, and the other is a three-dimensional gel sheet prepared by fusing cells by using a biological gel material. Among products in which the biological tissue membrane + cells are present, clinical studies have been conducted in artificial skin, artificial cornea, and other artificial tissue membranes. The human acellular amniotic membrane as a tissue engineering scaffold has the advantages of easy acquisition, good biocompatibility, degradability, low immunogenicity and the like, and is already used for manufacturing tissue engineering skin and tissue engineering cornea. It also has some disadvantages including poor strength, possible rejection after implantation, etc. At present, in the tissue engineering skin products on the market for treating skin burn and wounds in China, only allogeneic cells, namely 'skin on body', and autologous cells, namely 'PELNAC', are available.
Disclosure of Invention
In order to overcome the technical problems in the prior art, the invention innovatively provides a method for constructing a dermal fibroblast skin sheet and the dermal fibroblast skin sheet constructed by the method.
The invention provides a method for constructing a dermal fibroblast skin sheet, which comprises the steps of inoculating dermal fibroblasts amplified in vitro to an amniotic membrane tissue which is subjected to degreasing, decellularization and nucleic acid removal treatment for cell solidification, and constructing a composite structure of the dermal fibroblasts and the amniotic membrane tissue; then culturing in an amplification culture medium, and enriching high-purity cells under cell culture conditions to obtain the dermal fibroblast skin sheet. In the invention, the in-vitro large-scale production of adult somatic cells provides sufficient and high-purity living cells for the skin sheet, namely raw material production; and (3) immobilizing the raw material cells on a deactivated solidified structure, namely product production.
Wherein the dermal fibroblast is an adult dermal fibroblast (HDFB), preferably an FI-phenotype adult dermal fibroblast. Adult dermal fibroblasts can be classified into seven bayer phenotypes according to morphological and metabolic laws, wherein FI phenotype and FII phenotype are most common in the early stage of primary culture. The FI phenotype cells grow in a long fusiform arrangement, have higher proliferation speed, are the main cell types forming the thickest part of a dermis tissue layer, are cultured in vitro in a long fusiform shape, proliferate twice in about 12 hours, and secrete type I collagen to promote the proliferation of self and other cells of skin tissues. FII phenotype cells are epithelial, tightly growing, and slow in rate.
In the present invention, high purity adult dermal fibroblasts are obtained. In order to obtain high-purity FI phenotype fibroblasts, the invention proposes that purification must be carried out in the initial stage of primary culture, namely, in the invention, other phenotype cells are removed according to cell morphology by adopting a method of directly removing cells by micromanipulation in the third to fifth days of primary culture. In the invention, purified dermal fibroblasts are obtained by a method of micromanipulation and direct elimination in the initial stage of primary culture, and then FI phenotype cells with high purity can be obtained in the subsequent primary culture and passage. In contrast, the traditional method is to perform staining analysis after obtaining cells, distinguish phenotypes, and separate different cells by a stepwise digestion method, but the traditional method cannot completely eliminate the hybrid cells and takes about 7 days.
In the present invention, adult dermal fibroblasts are produced in high yield. In the invention, in the initial stage of primary culture, the concentration of FBS is improved, a high serum environment is utilized to promote the cells in the primary tissue to enter a culture container for adherent growth, the primary culture time is shortened, and the high-yield adult dermal fibroblasts are obtained. The initial stage of primary culture refers to the first 8 generations of cells starting to multiply in vitro.
In the present invention, inoculated cells having a high survival rate are obtained. In the inoculation process of the raw material cells, in order to improve the survival rate of the combined cells, collagen components are added in an amplification culture medium, so that the nutrition of the cells is increased, and a gel three-dimensional structure is provided, so that the cells grow on the skin sheet according to the original spindle shape. As cell density increases, collagen is depleted by the cells, but after maturation, the cells continue to secrete collagen for their own needs. In the present invention, the inoculation culture contains collagen. Preferably, the inoculated raw materials comprise dermal fibroblasts, collagen, and cell expansion media.
In the invention, the biological activity of the amniotic membrane tissue is removed: the invention carries out the nucleic acid removing process on the amniotic membrane tissue to remove the biological activity and reduce the rejection risk in use.
In a specific embodiment, the method for constructing the dermal fibroblast skin sheet according to the present invention comprises:
(1) combining the dermal fibroblast expanded in vitro with the amniotic tissue treated by decellularization, degreasing and nucleic acid removal for cell solidification to construct a composite structure of dermal fibroblast-amniotic tissue;
preferably, the dermal fibroblast expanded in vitro is combined with the amniotic membrane tissue which is processed by decellularization, degreasing, deproteinization and nucleic acid removal for cell solidification, so as to construct a composite structure of dermal fibroblast-amniotic membrane tissue
(2) Then culturing in a culture container under the condition of an amplification culture medium to obtain the dermal fibroblast skin sheet.
In the step (1), the dermal fibroblast is derived from a primary tissue of an adult dermal fibroblast. Preferably, the dermal fibroblast is a single phenotype type I phenotype cell (FI phenotype cell), and the cell has high proliferation speed and high content of secreted type I collagen.
In the present invention, adult dermal fibroblasts refer to dermal fibroblasts of a human individual (except a fetus) after birth.
Preferably, the primary tissue of adult dermal fibroblasts is derived from the human hind neck and can be obtained by perforation. Preferably, the volume of the primary tissue is 4-8 mm3. More preferably, the volume of the primary tissue is at least 4mm3。
Preferably, the primary tissue of dermal fibroblasts is cultured in the primary culture medium in a culture container for 2 to 3 days after being cut into small tissue pieces. Then, the primary tissue of adult dermal fibroblasts is digested, resuspended, diluted, inoculated, and expanded in vitro to obtain the dermal fibroblasts.
Wherein, the digestion, the heavy suspension and the dilution inoculation can be carried out once; when the yield is required to be increased (i.e., the yield is increased), or when the preparation is clinically used for multiple times, the preparation can be carried out for multiple times.
Wherein the digestion conditions are as follows: digesting for 2-3 minutes at 37 ℃ under the action of trypsin-EDTA.
Wherein the resuspension conditions are as follows: mechanically whipped in serum-containing solution at 37 ℃.
Wherein the dilution inoculation is performed in an amplification medium every 1cm2Area of culture inoculation 104And (4) carrying out subculture on the cells. Wherein, in the amplification culture medium, each generation needs to be cultured for 2-4 days.
Wherein the primary culture condition is that the culture is carried out at 37 ℃ and the carbon dioxide concentration is 5%. Preferably, the time of the primary culture is 5-8 days. Preferably, 3-5 days after primary culture, other phenotype cells are removed according to cell types, and high-purity adult dermal fibroblasts can be obtained in subsequent primary culture medium and subculture. Specifically, the method for screening dermal fibroblasts according to the present invention comprises: under microscopic examination, the contaminating cells were scraped off with a sterile instrument.
Wherein the primary cell culture medium comprises the following components in percentage by volume: 70-86% DMEM high-glucose medium, 12-18% Fetal Bovine Serum (FBS), 0.5-1% nonessential amino acids, 0.5-1% glutamine, and 1-10% streptomycin. In the initial stage of primary culture (3-5 days of the primary culture), the cells in the primary tissue are promoted to enter a culture container to grow adherent by improving the concentration of FBS and utilizing a high serum environment, so that the time of the primary culture is shortened.
Wherein the DMEM high-sugar medium is a commercial cell culture medium.
Wherein the non-essential amino acid is lysine.
The amplification medium contains the following components in percentage by volume: 84-92% of DMEM high-glucose medium, 7-15% of Fetal Bovine Serum (FBS), 0.5-1% of nonessential amino acid and 0.5-1% of glutamine.
In the present invention, the inoculation means: inoculating the cells in a culture vessel; the binding is to the amniotic membrane.
In the step (1), the dermal fibroblast after subculture is combined with an amniotic membrane tissue for cell immobilization. Wherein the raw materials for construction include dermal fibroblasts (raw material cells), collagen, cell amplification medium or physiological saline.
That is, collagen is added to dermal fibroblasts cultured in an amplification medium, and then the cells are bound to amniotic membrane tissue to be fixed; alternatively, collagen is added to physiological saline containing dermal fibroblasts, and then the mixture is bound to an amniotic membrane tissue to immobilize the cells. Preferably, the collagen is type I collagen. The collagen can increase the living cell density in the obtained composite structure of dermal fibroblast-amniotic membrane tissue and improve the inoculation survival rate of cells.
Wherein, the dermal fibroblast can be from autologous, non-autologous, xenogeneic and homologous dermal fibroblast domesticated in vitro.
Wherein the immobilization/cell immobilization is to inoculate the inoculation material (containing adult dermal fibroblast live cells) to an amniotic membrane tissue.
In a specific embodiment, the amniotic membrane tissue is sequentially subjected to the treatment steps of decellularization, degreasing, deproteinization and enucleation.
Wherein the main component of the cell-removing treatment solution is Triton X-100 (volume fraction is 0.1-0.5%).
Wherein the degreasing treatment solution mainly comprises normal saline and neutral lipase (the pH of the solution is 7.0-7.4, and the concentration of the neutral lipase is 5-10U/mL).
Wherein, the deproteinization treatment liquid mainly comprises physiological saline and alkaline trypsin.
Wherein, the nucleic acid removing treatment solution adopted in the nucleic acid removing treatment is a surface active solution, and the main components are nuclease and SDS (the PH of the solution is 7.0-7.4, and the mass concentration is 0.3-0.8%).
Wherein, the processes of decellularization, degreasing, deproteinization and nucleic acid removal are all carried out at 37 ℃, dynamic treatment is carried out, and the amnion is fixed.
Wherein, after the treatment process of each step is finished, the sterile normal saline is used for repeatedly cleaning.
Wherein, the amnion can be preserved for a long time by adopting a freezing method after being processed by the four steps, such as preservation at 4 ℃ in dark place for less than 30 days; wherein the frozen stock solution is replaced by centrifugation; the frozen stock solution is 50% serum frozen stock solution, the cooling method is programmed cooling, and the storage method is liquid nitrogen.
The purity test method of the cells in the dermal fibroblast-amniotic membrane tissue composite structure constructed by the method is an antibody method, CD73, CD90 and CD105 are incubated, the fluorescence of the cells is counted, and the purity of the fibroblasts in a cell product is calculated.
In a specific embodiment, the construction method comprises the following steps:
step 1): cleaning amniotic membrane tissue, and sequentially treating decellularized, degreased, deproteinized and denitrified by nucleic acid;
step 2): inoculating a dermal fibroblast living cell raw material into the amniotic membrane tissue, and carrying out cell solidification to construct a dermal fibroblast-amniotic membrane tissue composite structure;
step 3): amplifying and culturing in a culture container.
In another embodiment, the method of the present invention comprises extracting, screening, amplifying, identifying and inspecting, freezing, screening amnion, washing and sterilizing amnion, degreasing and decellularizing amnion, preserving amnion, solidifying cells, and culturing in semi-wet state to obtain the target product, i.e. autologous dermal fibroblast skin.
The invention also provides the autologous dermal fibroblast skin sheet obtained by the construction method.
The autologous dermal fibroblast skin sheet obtained by the construction method can be applied to clinical treatment of burn wounds, chronic diseases and large-area skin defects. The autologous dermal fibroblast skin sheet can be used for clinical requirements of burn wounds, large-area skin grafting and other skin defects which are urgently needed in large areas, provides low-rejection skin trauma auxiliary materials and increases clinical treatment methods; the autologous dermal fibroblast skin sheet obtained by the construction method can also be used for preparing skin care products, is used for preventing, eliminating and/or reducing wrinkles, and has wide application prospect.
Drawings
FIG. 1 shows a 100X in vitro cultured form of autologous dermal fibroblasts of the present invention.
FIG. 2 is a schematic representation of adult dermal fibroblasts of primary FI phenotype of the present invention, live cells being photographed at 40-fold magnification in a growth state.
Fig. 3 is a schematic representation of proliferation of adult dermal fibroblasts of the FI phenotype in collagen according to the present invention, live cells being microscopically photographed at 40-fold magnification in a growth state in the three-dimensional structure of collagen.
FIG. 4 is a graphical representation of the effect of clinical application of autologous dermal fibroblasts to treat facial augmentation; wherein, a represents the left lower eye circumference before injection; b represents the right lower eye circumference before injection; c represents the left lower eye circumference after injection; d represents the right lower eye circumference after injection.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and the accompanying drawings. The procedures, conditions, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art except for the contents specifically mentioned below, and the present invention is not particularly limited.
Example 1 construction of autologous dermal fibroblast skin sheet
In the construction method, the operation is as follows:
firstly, expanding dermal fibroblasts in vitro:
1) extraction:
a) in a sterilized state, from the user's nape line (the burn patient may take samples elsewhere), one to a plurality of thick skin tissues of 1.5mm diameter and 10mm depth are punched with a skin punch. And immediately filling the thick skin tissue into a tissue cleaning solution for sealing, refrigerating and storing in dark place for not more than 4 hours.
b) The formula of the tissue cleaning fluid is as follows: sterile water for injection, penicillin 1Ku/ml, streptomycin 1mg/ml, and inculwater (mycoplasma inhibitor) concentration 0.1% by volume.
c) Under aseptic experimental conditions, the epidermis of the thick skin tissue is partially cut off (3 mm below the epidermis), the subcutaneous fat part is cut off (yellow oil), and the fibrous area of the dermis area is reserved. On a sterile plate, repeatedly dipping the tissue in a tissue disinfectant for scrubbing by using a sterile cotton swab, soaking by using a newly configured sterile disinfectant, and replacing the disinfectant. And repeatedly soaking and cleaning.
d) The formula of the tissue disinfectant comprises: sterile water for injection, penicillin 1Ku/ml, streptomycin 1mg/ml, and inculwater (mycoplasma inhibitor) concentration 0.1% by volume.
e) The thick skin tissue is cut into small tissue blocks in a sterile plate, and the tissue blocks are tightly attached to the plate and cannot float. Adding primary cell culture solution, and adding CO at 37 deg.C2Culturing in 5% carbon dioxide incubator for 3 days.
f) The formula of the primary cell culture solution is as follows: DMEM high glucose medium + 15% fetal bovine serum + 1% non-essential amino acids + 1% glutamine + 1% streptomycin.
2) And (3) purification and screening:
g) the primary cell culture fluid was removed, sterile saline was added to the culture dish, and the primary cells were observed under microscopic field.
h) Observation analysis of each cell phenotype: wherein, the FI type is a slender fusiform cell, has moderate size and is arranged orderly; FII is an epithelial cell, a small polygon (similar to an epidermal cell), a large nucleus, and a tight arrangement; FIII is a large epithelial cell with a large nucleus.
i) In a sterile environment, all cells except the FI-type phenotype were carefully scraped. The culture dish is washed by sterile normal saline for a plurality of times to ensure that the hybrid cells are completely removed.
j) Adding the rest cells into cell amplification culture solution, and culturing in carbon dioxide incubator with CO2 concentration of 5% at 37 deg.C for 2-3 days until the cells are long fusiform, ordered arrangement and density of 90%.
3) Amplification:
the purified and screened primary tissue was digested with a trypsin-EDTA solution of non-animal origin. Digestion conditions were 37 ℃ for 2 to 3 minutes. Counting the number of the living cells of the cells obtained after digestion; then inoculating into primary culture medium for culture, and then performing amplification culture in amplification culture medium.
Wherein the primary culture medium comprises the following components in percentage by volume: 70-86% DMEM high-glucose medium, 12-18% Fetal Bovine Serum (FBS), 0.5-1% nonessential amino acids, 0.5-1% glutamine, and 1-10% streptomycin.
The amplification medium contains the following components in percentage by volume: 84-92% of DMEM high-glucose medium, 7-15% of Fetal Bovine Serum (FBS), 0.5-1% of nonessential amino acid and 0.5-1% of glutamine.
4) Identification and testing
Taking the solution obtained after the primary tissue is digested in the amplification in the step 3) as a cell-containing solution, and carrying out bacteria, mycoplasma and virus inspection. The aforementioned test results must be negative.
Taking the single cell suspension obtained in the step 3) for flow cytometry detection, wherein the detection antibody is CD: 73,90 and 105, and the positive rate is more than 95 percent.
5) Freezing and storing
The cells obtained after amplification are digested with pancreatin to obtain single cells, and then terminated with a serum-containing medium, and the subsequent construction of dermal fibroblast skin sheets can be performed.
Freezing the rest cells after treatment, and the specific steps are as follows: the cells were trypsinized into single cells and eluted twice with sterile physiological saline (centrifugation at 400g for 5 minutes). The centrifuged cells were resuspended in a freezing medium and dispensed at a density of 2 million/ml. Cooling and freezing at the speed of 1 deg.C/min.
The frozen cells can be stored for 2 to 5 years.
When the frozen cell tube is used again, the frozen cell tube is quickly melted in hot water (80 percent), transferred into sterile physiological saline at 4 ℃, and the frozen solution is replaced by a centrifugal method. After centrifugation, the cells are counted by viable cell counting method, and the viable cells can be inoculated.
Secondly, de-epithelializing amnion is used for constructing solid cells:
1) screening of amniotic Membrane tissue
And selecting the amniotic tissue without infectious diseases and amniotic abnormal pregnancy according to the virus test result of the blood sample provided by the donor.
2) Cleaning and disinfecting amnion tissue
Removing chorion from the newly delivered amnion, repeatedly washing with physiological saline to remove blood cells and other impurities, and retaining amnion tissue. The tissue comprises an epithelial cell layer, a basement membrane, and intact amniotic tissue without vascular stroma.
The obtained amnion is soaked and rinsed for 2 times by using medical alcohol with volume concentration of 75%, and then soaked and rinsed for 2 times by using tissue cleaning solution.
3) Decellularization-degreasing-deproteinization-enucleation sequential treatment of amniotic tissue
a) Decellularization
Soaking the amniotic membrane tissue in a lipase solution by using animal-derived lipase, incubating and digesting, wherein the digestion conditions are as follows: 37 ℃ and dynamic state. The tissue was transferred to sterile saline and residual cells of the epithelial layer were scraped off with a cell scraper.
The protease solution formula comprises: trypsin concentration 0.25% (mass volume concentration), EDTA concentration 0.005% (mass volume concentration), PH 8.4.
b) Degreasing
This step is used to remove cell membranes and membrane tissue of organelles, removing cellular genetic material.
Soaking amniotic membrane tissue in a lipase solution by using non-animal-derived lipase, incubating and digesting, wherein the digestion conditions are as follows: 37 ℃, 3 hours, dynamic.
Soaking the amniotic membrane tissue in a lipase solution by using a surfactant solution, incubating and digesting, wherein the incubating conditions are as follows: 37 ℃, 3 hours, dynamic.
The formula of the lipase solution comprises the following steps: lipase was prepared from sterile physiological saline at PH 9.0.
c) Deproteinization
The deproteinizing solution mainly comprises physiological saline and alkaline trypsin.
d) Denucleotiding
The amniotic tissue is soaked in a DNase solution and incubated for digestion at 37 ℃. After complete digestion, the amniotic membrane was rinsed 3 times with sterile physiological saline. DNA was removed by this step.
The nuclease solution formula comprises: sterile normal saline, the concentration of the DNase is 1000-.
4) Preservation of amniotic tissue
The amniotic tissue processed by the steps can be soaked in sterile physiological saline containing 10% of streptomycin, sealed at 4 ℃ and stored in a dark place. The effective period is 30 days.
Thirdly, constructing a composite structure of dermal fibroblast and amniotic membrane tissue, and culturing to obtain a target object, namely an autologous dermal fibroblast skin sheet:
1) constructing a composite structure of dermal fibroblast-amniotic tissue
The pieces of sheep membrane were laid flat in a culture vessel under aseptic conditions. Dissolving dermal fibroblast single cells in a cell amplification culture medium, adding type I collagen to prepare a dermal fibroblast single cell suspension, combining the dermal fibroblast single cell suspension with the amniotic membrane, and performing cell solidification to obtain a dermal fibroblast-amniotic membrane tissue composite structure of dermal fibroblast-collagen gel formed on the amniotic membrane.
2) Semi-wet culture
The composite structure tissue is laid in a culture container, the amnion faces downwards, and the dermal fibroblast-collagen gel faces upwards. And inoculating a small amount of amplification culture medium, allowing the liquid surface to submerge the gel layer, and culturing for 3 days to obtain the dermal fibroblast skin sheet.
The formula of the inoculation solution is as follows: normal saline or amplification medium, HDFB living cell suspension, type I collagen.
The cultured dermal fibroblast skin sheet can be directly applied to a wound. The main effective component in the skin sheet is fibroblast live cells, the cells can continuously secrete I-type collagen on the premise of clinging to the wound, nutrient substances are provided for the residual cells at the wound, and the exogenous live cells on the skin sheet are continuously proliferated and can be transferred to the wound to grow so that the wound is gradually healed.
The dermal fibroblast skin sheet constructed by the invention can promote the growth of the dermis and is mainly used for chronic wounds (diabetic ulcer, long-term bedsore, non-infectious long-term herpes and the like); it can also be used for treating II degree burn and below, and can also be used in healing stage after deep burn is treated by debridement in early stage.
Example 2 the autologous dermal fibroblast skin sheet test constructed in example 1:
1) content test of effective components
Sampling inspection is performed on batches of products. The content test method comprises the following steps of taking a skin piece with the area of 1cm2, rinsing the skin piece with physiological saline, and rinsing: rinse 2 times in sterile container. Digesting by using a skin piece digestive juice under the following digestion conditions: dynamic, fractionated digestion at 37 ℃ and collection, and termination of serum-containing solutions. The digests are collected and combined and may be concentrated by centrifugation.
The cells obtained by digestion collection and pooling were counted for the non-differential survival rate. The result of the content of the effective components is obtained.
The formula of the digestive juice comprises: trypsin, collagenase type I, physiological saline.
2) Active ingredient activity test
Sampling inspection is performed on batches of products. The content test method comprises the following steps of taking a skin piece with the area of 1cm2, rinsing the skin piece with physiological saline, and rinsing: rinse 2 times in sterile container. Digesting by using a skin piece digestive juice under the following digestion conditions: dynamic, fractionated digestion at 37 ℃ and collection, and termination of serum-containing solutions. The digests are collected and combined and may be concentrated by centrifugation.
And (4) collecting and combining the cells after digestion, and performing AOPI fluorescent dye staining counting. The activity result of the effective components is obtained.
The formula of the digestive juice comprises: trypsin, collagenase type I, physiological saline.
Fluorescent dye solution components: AO dye liquor; and (3) PI dye liquor.
3) Purity test of effective components
Microscopic examination is carried out to distinguish the growth form and growth cycle of the cells.
Sampling inspection is performed on batches of products. The content test method comprises the following steps of taking a skin piece with the area of 1cm2, rinsing the skin piece with physiological saline, and rinsing: rinse 2 times in sterile container. Digesting by using a skin piece digestive juice under the following digestion conditions: dynamic, fractionated digestion at 37 ℃ and collection, and termination of serum-containing solutions. The digests are collected and combined and may be concentrated by centrifugation.
Incubating the digested cells with antibodies comprising: CD73, CD90, CD 105. Incubation conditions were as follows: 4 ℃ for 1-4 hours.
The antibody detection method comprises the following steps: fluorescence counting method. And calculating the fluorescence concentration to obtain the purity of the effective components of the product.
The formula of the digestive juice comprises: trypsin, collagenase type I, physiological saline.
Example 3 in vitro production of adult autologous dermal fibroblasts.
In this example, the patient is a 33 year old female. The clinical indication is external canthus wrinkle grade I. The specific operation is as follows:
1) after biochemical test of blood, biochemical test of urine and virology test, patients are excluded from liver dysfunction, renal dysfunction and virus carrying risks and then are put into groups.
2) The skin collection site is the nape. The acquisition amount was 4mm 3. The collected skin tissue was stored in a tissue transfusion solution and transported for 2 hours at a temperature of 4 ℃.
3) The tissue was washed 2 times with tissue washes under sterile conditions, and the epidermis and subcutaneous adipose sites were removed with sterile instruments. And (3) performing aseptic cutting on the dermal tissue part, enabling the cut tissue to be tightly attached to a culture dish, adding a primary culture medium, and starting culture.
4) After 2 to 3 days of culture, primary dermal fibroblasts grew out in large quantities. Under microscopic observation, the non-FI phenotype cells are removed in a sterile environment, and the cells with high purity phenotype are reserved. As in fig. 1.
5) When the cells grow to 90% density, they are trypsinized, resuspended, diluted, inoculated and grown in amplification medium.
6) During subculture, a part of the cells were subjected to purity examination in this step. The purity of the fibroblasts in the product was checked by incubation with antibodies CD73, CD90, CD 105. The purity of the FI phenotype cells was checked by methionine assay.
7) The cells were expanded to 10 days within 28 days after the inoculation in step 5)7And (4) FI type living cells.
8) In the final cell digestion and collection step, all cells are digested with pancreatin, and the digested cells are terminated with a serum-containing medium. The medium in the cell suspension was replaced with physiological saline by centrifugation. Concentrating to obtain the living cell injection of normal saline.
9) And replacing the rest cells with frozen stock solution by centrifugation, performing programmed cooling, and permanently storing in liquid nitrogen for subsequent use.
Example 4
33 years old female face orbit outside and underside 1.5 grade wrinkle, autologous dermal fibroblast is injected and filled for beauty treatment
Collecting skin tissue
1) After the earlobe, 4mm of the sample was collected3Taking a dermis part of a whole-skin tissue with a certain volume, and carrying out adherent culture in a culture container;
2) after 3 days of primary culture, cells began to grow;
3) when the primary culture is carried out for 5 days, other cells except the FI phenotype are removed through micromanipulation;
4) performing primary culture for 7 days to obtain primary cultured dermal fibroblasts;
(II) production of autologous dermal fibroblasts
5) Performing primary digestion, dilution and amplification on the primarily cultured dermal fibroblasts, performing secondary digestion, dilution and amplification 14 days later, and simultaneously performing mycoplasma and virus detection and purity identification on raw material cells;
6) on day 21 of production, digestion, dilution and amplification are carried out for the third time;
7) on day 28 of production, raw material cells were collected; producing living cell preparation, and inspecting the quality of the preparation; clinical use (omitted); freezing and storing the rest cells;
8) the production is carried out once again at the later stage, and the clinical application is carried out once;
(III) clinical applications
The wrinkled area was rated on a 0.5 scale.
The autologous dermal fibroblasts of the present invention were prepared as an injection and directly injected into the orbital wrinkle region, and the results are shown in fig. 4, in which a represents the left lower periocular circumference before injection; b represents the right lower eye circumference before injection; c represents the left lower eye circumference after injection; d represents the lower eye circumference on the right side after injection, and the contrast shows that the dominant wrinkles disappear.
The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, which is set forth in the following claims.
Claims (6)
1. A method for constructing a dermal fibroblast skin sheet is characterized by comprising the following steps:
step (1): combining the dermal fibroblast expanded in vitro with the amniotic tissue treated by decellularization, degreasing, deproteinization and nucleic acid removal for cell solidification to construct a composite structure of dermal fibroblast-amniotic tissue; the dermal fibroblast is an FI phenotype adult dermal fibroblast;
the primary tissue of dermal fibroblasts is cultured in a primary culture medium containing the following components in volume percent: 70% -86% of a DMEM high-glucose medium, 12% -18% of fetal bovine serum, 0.5% -1% of nonessential amino acids, 0.5% -1% of glutamine, and 1% -10% of streptomycin;
wherein the primary culture condition of the dermal fibroblasts is that the primary culture is carried out at the temperature of 37 ℃ and the carbon dioxide concentration of 5 percent;
the dermal fibroblasts are purified by a method of micromanipulation and direct elimination in the initial primary culture stage;
the cell-removing treatment solution comprises Triton X-100 with the volume fraction of 0.1-0.5%;
the degreasing treatment liquid comprises normal saline and neutral lipase, the pH value is 7.0-7.4, and the concentration of the neutral lipase is 5-10U/mL;
the deproteinizing solution comprises normal saline and alkaline trypsin;
the nucleic acid removing treatment solution comprises nuclease and SDS, the pH of the solution is 7.0-7.4, and the mass concentration is 0.3-0.8%; the treatment processes of decellularization, degreasing, deproteinization and nucleic acid removal are all carried out at 37 ℃, dynamic treatment is carried out, and the amnion is fixed;
wherein the non-essential amino acid is lysine;
the single cell suspension of the FI phenotype adult dermal fibroblasts is subjected to flow cytometry, and detection antibodies are CD73, CD90 and CD105, and the positive rate is more than 95%;
step (2): culturing in an amplification culture medium to obtain the dermal fibroblast skin sheet;
the amplification medium contains the following components in percentage by volume: 84% -92% of DMEM high-glucose medium, 7% -15% of Fetal Bovine Serum (FBS), 0.5% -1% of nonessential amino acid and 0.5% -1% of glutamine;
the dermal fibroblast skin sheet is obtained by semi-wet culture of a composite structure of dermal fibroblast and amniotic tissue, and comprises the following specific steps:
constructing a composite structure of dermal fibroblast-amniotic tissue
Under aseptic condition, laying the sheep membrane in a culture container; dissolving dermal fibroblast single cells in a cell amplification culture medium, adding type I collagen to prepare a dermal fibroblast single cell suspension, combining the dermal fibroblast single cell suspension with an amniotic membrane, and performing cell solidification to obtain a dermal fibroblast-amniotic membrane tissue composite structure of dermal fibroblast-collagen gel formed on the amniotic membrane;
semi-wet culture
The composite structure tissue is flatly laid in a culture container, the amnion faces downwards, and the dermal fibroblast-collagen gel faces upwards; inoculating the amplification culture medium, allowing the liquid surface to submerge the gel layer, and culturing for 3 days to obtain the dermal fibroblast skin sheet.
2. The method of claim 1, wherein the primary tissue of dermal fibroblasts is cultured in a primary culture medium; then carrying out the in vitro amplification by digestion-resuspension-dilution inoculation; wherein the digestion conditions are as follows: digesting for 2-3 minutes at 37 ℃ under the action of trypsin-EDTA; the resuspension conditions were: mechanically beating in serum-containing solution at 37 deg.C; the dilution inoculation is carried out in an amplification medium at a concentration of 1cm2Area of culture inoculation 104And (4) carrying out subculture on the cells.
3. The method of claim 1, wherein the dermal fibroblast is bound to the amniotic membrane tissue for cell solidification by seeding culture; the construction raw materials for inoculation culture comprise dermal fibroblasts, collagen, a cell amplification culture medium or normal saline.
4. A dermal fibroblast skin sheet prepared by the method of claim 1.
5. Use of a dermal fibroblast skin sheet according to claim 4 in the manufacture of a product for the treatment of burn wounds, chronic diseases or skin defects, slow-wound burns, or in the manufacture of a product for skin grafting.
6. The use of claim 5, wherein the dermal fibroblast skin sheet is for preventing, eliminating and/or reducing wrinkles.
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