CN113025654A - Construction method of human induced pluripotent stem cell bank - Google Patents
Construction method of human induced pluripotent stem cell bank Download PDFInfo
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Abstract
The invention discloses a construction method of a human induced pluripotent stem cell bank, belonging to the technical field of preparation of induced pluripotent stem cells. The method of the invention establishes induced pluripotent stem cells by reprogramming skin fibroblasts, and induces passage and cryopreservation of the pluripotent stem cells; establishing a database of induced pluripotent stem cells. The method can rapidly and efficiently reprogram the skin fibroblasts into the induced pluripotent stem cells in a short time, avoids the addition of exogenous genes, can ensure the cell activity and the proliferation capacity, has stable passage, high expression of stem cell specific protein, good multidirectional differentiation capacity and long-term storage. Serum-free culture medium and serum substitute are used for preparing the freezing medium, so that the influence of non-human-derived substances on cell surface markers and purity is avoided, and safe and nontoxic seed cells are provided for the stem cells derived from later donors and the use of stem cell preparations.
Description
Technical Field
The invention relates to a construction method of a human induced pluripotent stem cell bank, belonging to the technical field of preparation of induced pluripotent stem cells.
Background
Induced Pluripotent Stem Cells (ipscs) were originally one cell type similar to embryonic Stem Cells obtained by transferring a combination of 4 transcription factors OSKM (Oct4, Sox2, Klf4 and c-Myc) into already differentiated somatic Cells using a viral vector in 2006 by Yamanaka, a japanese scientist, and reprogramming the Cells; the iPSC has the ability of proliferation and multiple differentiation, and can be derived from somatic cells of a donor, so that the iPSC can not cause immunological rejection in clinical application, and can avoid the medical ethical problems brought by embryonic stem cells, thereby playing an important role in various aspects such as disease modeling, drug discovery, cell therapy and the like, and promoting the development of subjects such as cytobiology, regenerative medicine and the like.
However, there are many reprogramming systems for inducing pluripotent stem cells, such as reprogramming methods by integrating a viral vector transfer system, but some reports indicate that the reactivation of viral transgenes and their residual activity in the production of i PSCs may alter the development process of cells due to the integration of exogenous genes, may lead to the formation of chimeric animal tumors, and have great limitations on stem cells in the later stage and stem cell preparations. The reprogramming efficiency of the integrated non-viral vector is extremely low, the requirements on clinical samples are difficult to obtain and samples of some intractable diseases are extremely high, and a small amount of somatic cells cannot be reprogrammed.
Disclosure of Invention
In view of the defects of the prior art, the technical problems to be solved by the invention are as follows: the induced pluripotent stem cells generated by reprogramming by adopting the prior art have the problems of low efficiency, influence on normal genome and karyotype of cells by transcription factor integration, poor cell activity, easy autonomous differentiation and containing exogenous components.
The invention aims to provide a culture method for reprogramming to establish induced pluripotent stem cells, which constructs a human induced pluripotent stem cell bank in a safer and more efficient manner through the processes of preparation, purification, amplification, identification and cryopreservation; the method can rapidly and efficiently reprogram the skin fibroblasts into induced pluripotent stem cells in a short time, can ensure the cell viability and the proliferation capacity, has stable passage, high expression of stem cell specific protein, good multidirectional differentiation capacity and long-term storage.
The invention is realized by the following technical scheme: a method for constructing a human induced pluripotent stem cell bank specifically comprises the following steps:
(1) reprogramming skin fibroblasts to establish induced pluripotent stem cells, wherein the reprogramming system is a non-integration type virus system, the selected reprogramming virus is Sendai virus, and transcription factors contained in the Sendai virus are c-Myc, SeV, KOS and Klf 4.
(2) And (5) inducing passage and freezing storage of the pluripotent stem cells.
(3) Establishing a database of induced pluripotent stem cells: the database comprises six items of data: cell activity detection results, cell infection detection results, genetic disease detection results, cell carcinogenicity detection results, HLA-ABC/DR match results, cell sources, and establishing correlation with cryopreserved cells.
Preferably, the reprogramming of skin fibroblasts to establish induced pluripotent stem cells of the invention specifically comprises the following steps:
firstly, collecting skin tissues, separating fibroblasts from the obtained skin tissues by adopting an enzyme digestion adherence method, and carrying out passage, cryopreservation and related identification on the fibroblasts;
② two days before virus transfection, third generation of frozen fibroblasts are inoculated into at least two holes of a six-hole plate, and the cell density on the day of transduction reaches 2X 10 per hole5-3×105；
③ adding the non-integrative virus vector into the preheated fibroblast culture medium, adding the culture medium into the fibroblasts, then, the cells are cultured under the condition of 37 ℃ and 5 percent CO2Incubating overnight in the incubator;
fourthly, after the virus factors are transferred into the culture medium for 24 hours, the culture medium is replaced to remove the virus, then the fibroblast culture medium is used for continuously culturing the cells for 6 days, and the liquid is replaced every other day;
fifthly, on the 7 th day, coating the six-hole plate with 1ml of diluted Geltrex Membrane Mtrix (Gibco) or Matrigel Matrix (Corning) at 37 ℃ for 1 h;
sixthly, digesting and centrifugally collecting cells by 0.5ml of 0.05-0.25 percent EDTA trypsin, and according to the proportion of 5 multiplied by 10 per hole4-10×104The cells are inoculated on a six-well plate coated by a matrix according to the density; adding fibroblast culture medium, 5% CO at 37 deg.C2Incubating in an incubator overnight;
seventhly, after 24 hours, replacing the fibroblast culture medium with an Essential 8 complete culture medium, and replacing the culture medium once a day; observing the appearance of induced pluripotent stem cell clones every other day under a microscope from day 8;
the colonies are usually observed to form on day 12, after 3-4 weeks the colonies grow to the appropriate size, undifferentiated stem cell colonies are manually picked, scored as first generation (P1) and transferred to Geltrex Membrane Mtrix (Gibco) coated culture dishes for further expansion.
Preferably, the culture medium used in the fibroblast culture process of the present invention is: DMEM basal medium + 10% fetal bovine serum + 1% NEAA + 1% double antibody.
Preferably, the specific process of the passage of the induced pluripotent stem cells of the invention is as follows: when the Cell fusion degree reaches 80% -90%, using 0.5ml of Gentle Cell discovery Reagent (STEMCELL), digesting for 5-8min at 37 ℃, observing the gradual Dissociation of Cell clones under a microscope, gradually loosening Cell connection, removing digestive juice, adding 0.5ml of PBS to slightly clean residual digestive juice, removing PBS, adding Essential 8 complete culture medium, slightly shaking the culture dish, transferring the Cell clones which fall from the bottom of the culture dish into clones of 5-10 Cell masses, transferring the Cell clones into a new six-well plate, adding 2ml of Essential 8 complete culture medium, and continuing culture.
Preferably, the specific process of cryopreserving the induced pluripotent stem cells comprises the following steps: when the Cell fusion degree of passage is 80% -90%, using 0.5ml of Gentle Cell Dissociation Reagent (STEMCELL), digesting for 5-8min at 37 ℃, observing the gradual Dissociation of Cell clones under a microscope, gradually loosening Cell junctions, removing digestive juice, adding 0.5ml of PBS, slightly washing residual digestive juice, removing PBS, adding 1ml of freezing solution, and preparing the freezing solution by KnockOutTM Serum Replacement (Gibco) + 10% DMSO (Sigma); gently shaking the culture dish, collecting cells in a 1.8ml cryopreservation tube, placing in a programmed cryopreservation box (Corning), and placing in a refrigerator at-80 deg.C; after 24 hours, the cells were transferred from-80 ℃ to liquid nitrogen for storage.
Preferably, the serum-free medium used in the induced pluripotent stem cell culture process is Essential 8TMMedium (Gibco) or StemFlexTM Medium(Gibco)。
The fibroblasts of the present invention include, but are not limited to, skin fibroblasts of human and mammalian origin.
Preferably, the database of human induced pluripotent stem cells of the present invention comprises the following contents:
(1) and (3) detecting the activity of the cells: counting after trypan blue staining, recording the cell number of the cryopreserved cells before and after cryopreservation, and drawing a growth curve by using an MTT method.
(2) Detection of cell infection: detecting whether the cells are polluted by bacteria, fungi and mycoplasma by using a small amount of cell culture, and detecting whether animal-derived substances exist; and detecting the presence of parvovirus.
(3) Detection of genetic diseases: and detecting whether the frozen cells have genetic diseases or not by using a molecular genetics method.
(4) And (3) detecting the carcinogenicity of the cells: the carcinogenicity of the cells was detected by the formation of soft agar clones and the activity of experimental telomerase and recorded.
(5) HLA-ABC/DR match: HLA-ABC/DR phenotype was tested and documented.
(6) Recording of cell origin: record the details of the supplier and record in case.
The induced pluripotent stem cell line established by the invention has strict quality control on cells, ensures the safety of the cells, is cultured by using a serum-free culture medium in the culture process, does not contain exogenous components, and provides safe seed cells for later clinical treatment.
Compared with the prior art, the technical scheme of the invention has the following advantages:
(1) the invention provides a non-integrative virus for reprogramming fibroblasts, which avoids the addition of exogenous genes, uses a serum-free culture medium and a serum substitute to prepare a freezing medium, avoids the influence of non-human substances on cell surface markers and purity, and provides safe and non-toxic seed cells for the use of stem cells from later donors and stem cell preparations.
(2) According to the method, the pluripotent stem cells are induced by digestion with mild digestive juice, so that the damage of enzymatic digestion to the stem cells is avoided, and the cell activity and the proliferation capacity are improved.
(3) The method avoids the damage of centrifugation to cells, mildly carries out passage cryopreservation on the cells, better ensures the cell activity and the proliferation capacity, reduces the autonomous differentiation rate of the cells, improves the cell purity and ensures the high expression of the stem cell specific protein.
(4) The method of the invention detects various genetic diseases and infectious diseases from sample sources, has clear sample sources and comprehensive information, and ensures cell safety and sample library safety
(5) The method of the invention detects various microorganisms such as mycoplasma, fungi, viruses and the like which induce the pluripotent stem cells, strictly controls the quality and ensures the cell safety.
Drawings
FIG. 1 is the morphology of the normally grown induced pluripotent stem cells of example 1.
FIG. 2 shows the expression of the transcribed genome of the virus of example 1.
FIG. 3 is the identification of short tandem repeats of the induced pluripotent stem cells in example 1.
FIG. 4 is the karyotype identification of fibroblasts and induced pluripotent stem cells in example 1.
FIG. 5 shows immunofluorescence identification of induced pluripotent stem cell surface markers in example 1.
FIG. 6 is a cytoflow assay of induced pluripotent stem cell surface markers according to example 1.
FIG. 7 is an immunofluorescence assay for inducing differentiation of the three germ layers of pluripotent stem cells in example 1.
Detailed Description
The present invention will be described in further detail with reference to the following examples, but the scope of the present invention is not limited to the above description.
The serum-free human mesenchymal stem cell culture medium and the cell digestive juice of the reagent used in the invention are all commercial products.
In the embodiment of the invention, the seed cells are derived from human skin fibroblasts.
Example 1
Reprogramming skin fibroblasts to establish induced pluripotent stem cells, specifically comprising the following steps:
(1) signing an informed consent with a tissue donor, collecting the skin tissue of the waist of the donor with the depth of 5mm by using a skin sampler with the diameter of 4mm under the aseptic condition, simultaneously collecting 10mL of blood sample of the donor, and detecting the blood type, HA-ABC/DR match type and infectious diseases (hepatitis B, hepatitis C, cytomegalovirus, EB virus, syphilis and AIDS virus) of the donor to ensure that all indexes are normal.
(2) And (3) separating the fibroblasts from the obtained skin tissue by adopting an enzyme digestion adherence method, and carrying out passage, cryopreservation and related identification on the fibroblasts.
(3) Two days before virus transfection, the frozen third generation fibroblasts were thawed at a suitable density and seeded in at least two wells of a six-well plate to achieve a cell density of 2X 10 cells per well on the day of transduction5-3×105。
(4) Adding into 1ml of fibroblast culture medium preheated at 37 deg.C
2.0 Sendai reprogramming vector, add culture medium to fibroblasts, then cells at 37 deg.C, 5% CO2Incubate overnight in the incubator.
(5) After the virus factor is transferred for 24h, the culture medium is changed to remove the virus, and then the cells are continuously cultured for 6 days by using the fibroblast culture medium, and the liquid is changed every other day.
(6) On day 7, the hexawell plates were coated with 1ml of diluted Geltrex Membrane Mtrix (Gibco) or Matrigel Matrix (Corning) for 1h at 37 ℃.
(7) The cells were collected by centrifugation digested with 0.5ml of 0.05% -0.25% EDTA trypsin and centrifuged at 5X 10 per well4-10×104The cells are inoculated on a six-well plate coated by a matrix according to the density; adding fibroblast culture medium, 5% CO at 37 deg.C2Incubate overnight in the incubator.
(8) After 24 hours, the fibroblast medium was changed to Essential 8 complete medium (Gibco) once a day; the appearance of induced pluripotent stem cell clones was observed under a microscope every other day from day 8.
(9) Colony formation is typically observed on day 12, after 3-4 weeks the colonies grow to the appropriate size, undifferentiated stem cell clones are manually picked, scored as first generation (P1), and transferred to petri dishes coated with Geltrex Membrane Mtrix (Gibco) for further expansion.
Passage and cryopreservation of induced pluripotent stem cells, and the specific operation steps are as follows:
(1) passage of induced pluripotent stem cells:
when the Cell fusion degree reaches 80% -90%, using 0.5ml of Gentle Cell Dissociation Reagent (STEMCELL), digesting for 5-8min at 37 ℃, observing the gradual Dissociation of Cell clones under a microscope, gradually loosening Cell connection, removing digestive juice, adding 0.5ml of PBS to slightly clean residual digestive juice, removing PBS, adding Essential 8 complete culture medium, slightly shaking the culture dish, removing the Cell clones which fall from the bottom of the culture dish into 5-10 Cell blocks, transferring the Cell blocks into a new six-well plate, adding 2ml of Essential 8 complete culture medium, and continuing culture.
(2) Cryopreservation of induced pluripotent stem cells:
when the Cell confluence at passage is 80% -90%, using 0.5ml of Gentle Cell Dissociation Reagent (STEMCELL), digesting for 5-8min at 37 ℃, observing the gradual Dissociation of Cell clones under a microscope, gradually loosening Cell junctions, removing digestive juice, adding 0.5ml of PBS to gently wash residual digestive juice, removing PBS, adding 1ml of freezing solution, and preparing the freezing solution by KnockOutTM Serum Replacement (Gibco) + 10% DMSO (Sigma). Gently shaking the culture dish, collecting cells in a 1.8ml cryopreservation tube, placing in a programmed cryopreservation box (Corning), and placing in a refrigerator at-80 deg.C; after 24 hours, the cells were transferred from-80 ℃ to liquid nitrogen for storage.
FIG. 1 shows induced pluripotent stem cells that grow normally after passage, are round in shape, have a large nuclear-to-cytoplasmic ratio, have multiple nuclei, are in a clonal state in a culture dish, and have a compact and uniform clone with clear boundaries from the periphery.
The identification of the induced pluripotent stem cell transcription factor genome prepared in the embodiment specifically comprises the following operation steps:
(1) extraction of cellular RNA
(ii) digestion and centrifugation with Gentle Cell Dissociation Reagent (Stemcell) 105-106The P3-P8 iPSC and the iPSC after P10 were put in a 1ml centrifuge tube.
② adding cell lysate to lyse the cells, incubating for 3 minutes at 15-30 ℃, and centrifuging for 10 minutes at 12000rpm under 4 ℃.
③ adding 0.2ml of chloroform into each 1ml of cell lysate, violently shaking for 15s, then incubating for 3 minutes at room temperature, centrifuging for 10 minutes at 12000rpm, transferring the upper aqueous phase into a new centrifuge tube, and then adding 0.5 times of volume of absolute ethyl alcohol.
Fourthly, the mixture is reversed and evenly mixed and then is transferred into an adsorption column; centrifuging at 12000rpm for 45s, removing waste liquid, adding 500 μ l rinsing solution, cleaning twice, placing the adsorption column into a new centrifuge tube, adding 50-100 μ l RNase-free water, and eluting RNA. The concentration of RNA was then measured using a Nanodrop.
(2) The extracted RNA is reverse transcribed into cDNA.
A100. mu.l centrifuge tube was prepared, to which 50 ng-5. mu.g of RNA, 3. mu.l of RT Enzyme Mix and 7. mu.l of RT Reaction Mix were added, followed by supplementation to 20. mu.l with dd water; then incubating at 45 ℃ for 30min, and inactivating at 85 ℃ for 5min to obtain cDNA.
(3) PCR amplification
Preparing a 100 mu l centrifuge tube, and adding 1 mu l template cDNA, 1 mu l upstream primer, 1 mu l downstream primer, 12.5 mu l 2 XTaq Plus PCR Master Mix, ddH2O to a 25 mu l reaction system in sequence; pre-denaturation at 94 ℃ for 3min, PCR at 94 ℃ for 30s, PCR at 55 ℃ for 30s, and PCR at 72 ℃ for 1min for 35 cycles, and then preservation at 4 ℃ after 5min at 72 ℃.
Four sets of transcription factor primers
(4) Agarose gel electrophoresis and development
Diluting 10ml 50 × TAE to 500ml, pouring 450ml TAE into electrophoresis tank; putting 40ml of 1 XTAE solution into a conical flask, adding 0.8mg of agarose, heating in a microwave oven for 2min, fully dissolving, slightly cooling, adding 0.8 mu l of nucleic acid dye, stirring uniformly, pouring the agarose solution into a plate, inserting a tooth comb, cooling, putting into an electrophoresis tank, and pulling out the comb; adding 5 μ l sample, performing electrophoresis at constant voltage of 75V-100V for 30min-1h, and detecting with imaging instrument.
In this example, P3 and P13 were used in place of iPSC for detection, and as shown in FIG. 2, four transcription factors were detected in P3 (FIG. 2.A) and not in P13 (FIG. 2. B).
The analysis of the method of the embodiment shows that the method can successfully transfer the four transcription factors into the cells and express the transcription factors, and after a period of culture, the transcription factors disappear from the induced pluripotent stem cells and the transcriptome of the cells is not influenced.
The induced pluripotent stem cells prepared in the embodiment are subjected to Short Tandem Repeat (STR) identification, and the specific operation steps are as follows:
(1) extraction of genomic DNA
(2) STR detection
Mu.l of DNA samples were taken for multiplex amplification of 21 loci, and PCR products were separated by capillary electrophoresis using ABI PRISM 3500xL Gene Analyzer and analyzed using GeneMapper1 ID-X software v 1.5.
FIG. 3 is the result of STR identification of maternally derived fibroblasts and iPSCs, showing that both cell genotypes are identical at the 21 selected loci; as can be seen from the analysis by the method of the present embodiment, the human induced pluripotent stem cells prepared by the method of the present invention have the same source as fibroblasts.
The induced pluripotent stem cell karyotype identification prepared in the embodiment has the following specific operation steps:
to verify whether reprogramming has an effect on cell karyotypes, ipscs were karyotyped. The ipscs were inoculated into T25 flasks and when they grew to logarithmic growth phase (confluency 70% -80%), colchicine was added thereto to a final concentration of 0.2 μ g/ml for two hours of culture. Collecting cell suspension, adding low-permeability liquid dropwise, and placing in 37 ℃ water bath for 15 minutes. Adding about 200 μ L of freshly prepared fixative, pre-fixing for 2-3 min, centrifuging at 800rpm for 8min, removing supernatant, adding 3-5 ml of fixative dropwise to resuspend cells to obtain single cell suspension, fixing for 40 min, centrifuging at 800rpm for 8min, and repeating the fixing for 2-3 times. Then removing supernatant, leaving 0.5-1mL of stationary liquid, mixing the cells into single cell suspension, sucking the cell suspension, dripping the cell suspension onto a precooled glass slide, and drying at room temperature. Taking out the slide, and dropwise adding Giemsa working solution to complete covering, wherein the dyeing is carried out for 20-30 minutes; washing with tap water to stop dyeing, drying at 35 deg.C, and sealing with sealing agent. Baking the prepared undyed glass slide in an oven at 70 ℃ for 2 hours for aging treatment, placing the glass slide in 0.025% pancreatin working solution for 2-3 minutes, slightly shaking, rinsing the glass slide in PBS, and sealing after Giemsa staining. Selecting a visual field, collecting a photo, analyzing by a Videotest chromosome karyotype analysis software, and obtaining a result.
The results are shown in FIG. 4, where the karyotype of the induced pluripotent stem cells was 46, xx, indicating that the nuclear type of the cells was not altered by the transcription factor.
The identification of the induced pluripotent stem cell marker protein expression prepared in this example includes the following specific operation steps:
(1) cell immunofluorescence detection induced pluripotent stem cell marker protein:
after the cell fusion degree reaches 30-50%, absorbing and removing the culture solution, and washing for 5min each time for 3 times by using 0.01M PBS solution;
dropping 4% paraformaldehyde solution, and fixing at room temperature for 30 min;
removing paraformaldehyde fixing solution by suction, washing for 3 times with 0.01M PBS solution, 5min each time, and adding 0.01M PBS diluted 0.3% Triton-X100 membrane for 15 min;
fourthly, removing Triton-X100 by suction, washing for 3 times by 0.01M PBS solution, each time for 5min, adding 10 percent goat serum, and sealing for 30min at room temperature;
fifthly, abandoning the sealing liquid, adding the diluted SOX2.OCT4.NANOG. TRA1-60.SSEA4 polyclonal antibody (1:500) into the cells, and incubating overnight in a refrigerator at 4 ℃.
Sixthly, after the primary antibody is incubated overnight, washing is carried out for 3 times by using 0.01M PBS solution, and each washing is carried out for 5 min.
And (c) incubating FITC fluorescent labeled secondary antibody (1:50) for 2h at room temperature in a dark place.
The second antibody was aspirated and washed 3 times with 0.01M PBS solution for 5min each time.
Ninthly, DAPI (1:1000 dilution) is added dropwise for staining the nucleus, and incubation is carried out for 10min in the dark at room temperature.
The red spot was washed 3 times with 0.01M PBS solution for 5min each time. The photographs were observed by fluorescence microscopy.
(2) Detecting the expression of the induced pluripotent stem cell marker protein by using the cell flow method:
digesting and collecting iPSC by using Gentle Cell Dissociation Regent (stem Cell), counting cells, taking 105-106 cells from each staining sample in a 15ml centrifuge tube, shaking and uniformly mixing the cells for 3s by using 1ml of freshly prepared 1 xFix/Perm Buffer (BD) working solution, incubating the cells for 40-50min at the temperature of 2-8 ℃ in the dark, and directly adding 1ml of 1 xPerm/Wash Buffer into the cells fixed with the rupture membrane. Centrifuging at 2-8 deg.C for 6min at 350g, discarding supernatant, adding 2ml of 1 XPerm/Wash Buffer, and centrifuging and cleaning once. Resuspending the cells with 80-100. mu.l of 1 XPerm/Wash Buffer, adding specific antibody (PE-SOX2, Alexa Flour 647-OCT3/4, PerCP-Cy5.5-SSEA4, FITC-TRA 1-60; BD) and nonspecific isotype control staining, vortexing and mixing, incubating at 2-8 ℃ in the dark for 40-50 min; after washing the cells twice with 2ml of 1 XPerm/Wash Buffer, 350. mu.l of Wash Buffer was added to resuspend the cells, which were detected and analyzed by flow cytometry.
Fig. 5 and fig. 6 show the results of cellular immunofluorescence and cytometric assay, respectively, and it can be seen from the figure that induced pluripotent stem cells are strongly positive for the protein marker of sox2.oct4.nanog.tra1-60.SSEA4 (fig. 5), and the positive rate of flow assay of each index is: 98.86 percent of SOX 2; 96.65% of OCT 4; 99.76 percent of SSEA 4; TRA1-60: 90.02%, all above 90% (FIG. 6).
As can be seen from the analysis by the method of the present embodiment, the induced pluripotent stem cell prepared by the method of the present invention conforms to the general characteristics of embryonic stem cells, is in an undifferentiated growth state, and has proliferation capacity.
In this example, the specific operation steps for preparing the induced pluripotent stem cell for identifying the multi-differentiation capacity are as follows:
the induced pluripotent stem cells were collected by digestion with a Gentle Cell differentiation reagent (stem Cell), the cells were resuspended in DMEM/F12, and counted by trypan blue direct Cell counting method, and the cells were applied to an appropriate density (ectoderm 5X 10)4Mesoderm and endoderm 1 × 105) Seeded on matrix-coated 12-well plates, to which ectodermal, mesodermal and endodermal differentiation media were added, respectively, and the fluids were changed daily. Mesodermal and endodermal cells mature after 5 consecutive days of fluid exchange, while ectodermal cells are also available by day 7. Cells were then fixed with 4% paraformaldehyde and immunofluorescent stained with a three germ layer antibody (ectoderm: TUJ, mesoderm: α -SMA, endoderm: FOXA 2).
As a result, as shown in fig. 7, the differentiated cells were strongly positive for the antibody of the three germ layers. As can be seen from the analysis by the method of the present example, the induced pluripotent stem cell prepared by the method of the present invention can be successfully induced and differentiated towards ectoderm, mesoderm and endoderm, and has a multi-differentiation potential.
Example 2
Establishment of human induced pluripotent stem cell database
(1) And (3) detecting the activity of the cells: counting after trypan blue staining, recording the cell number of the cryopreserved cells before and after cryopreservation, and drawing a growth curve by using an MTT method.
(2) Detection of cell infection: detecting whether the cells are polluted by bacteria, fungi and mycoplasma by using a small amount of cell culture, and detecting whether animal-derived substances exist; and detecting the presence or absence of parvovirus,
(3) detection of genetic diseases: and detecting whether the frozen cells have genetic diseases or not by using a molecular genetics method.
(4) And (3) detecting the carcinogenicity of the cells: the carcinogenicity of the cells was detected by soft agar colony formation and experimental telomerase activity and recorded.
(5) HLA-ABC/DR match: HLA-ABC/DR phenotype was tested and documented.
(6) Recording of cell origin: record the details of the supplier and record in case.
(7) Establishing an induced pluripotent stem cell database: after the normal induced pluripotent stem cells are preserved, establishing a database of the induced pluripotent stem cells, wherein the database comprises the first six items of data, and establishing association with the cryopreserved cells.
The method uses human skin fibroblasts, non-integrative viruses to transfect cells, uses a serum-free culture medium to culture induced pluripotent stem cells, uses mild digestive juice to digest the induced pluripotent stem cells, uses serum to replace cryopreserved cells, establishes a database of the human induced pluripotent stem cells, and associates the database with the cryopreserved cells; the method disclosed by the invention has the advantages that the efficiency of generating the induced pluripotent stem cells is high, no exogenous gene or exogenous substance is contained, and the proliferation capacity and the differentiation capacity are high; in addition, the induced pluripotent stem cell bank can be built for all people, can be used for clinical and basic research after personal permission, and can provide autologous stem cells and stem cell preparations for some clinically refractory disease patients.
Claims (6)
1. A method for constructing a human induced pluripotent stem cell bank is characterized by comprising the following steps:
(1) reprogramming skin fibroblasts to establish induced pluripotent stem cells, wherein the reprogramming system is a non-integrative virus system, the selected reprogramming virus is Sendai virus, and transcription factors contained in the Sendai virus are c-Myc, SeV, KOS and Klf 4;
(2) inducing passage and freezing storage of the pluripotent stem cells;
(3) establishing a database of induced pluripotent stem cells: the database comprises six items of data: cell activity detection results, cell infection detection results, genetic disease detection results, cell carcinogenicity detection results, HLA-ABC/DR match results, cell sources, and establishing correlation with cryopreserved cells.
2. The method of constructing a bank of human induced pluripotent stem cells according to claim 1, wherein the method comprises: the reprogramming skin fibroblast to establish the induced pluripotent stem cell specifically comprises the following steps:
firstly, collecting skin tissues, separating fibroblasts from the obtained skin tissues by adopting an enzyme digestion adherence method, and carrying out passage, cryopreservation and related identification on the fibroblasts;
② two days before virus transfection, third generation of frozen fibroblasts are inoculated into at least two holes of a six-hole plate, and the cell density on the day of transduction reaches 2X 10 per hole5-3×105；
③ adding the non-integrative virus vector into the preheated fibroblast culture medium, adding the culture medium into the fibroblast, then the cells are cultured at 37 ℃ and 5 percent CO2Incubating overnight in the incubator;
fourthly, after the virus factors are transferred into the culture medium for 24 hours, the culture medium is replaced to remove the virus, then the fibroblast culture medium is used for continuously culturing the cells for 6 days, and the liquid is replaced every other day;
fifthly, on day 7, coating the hexawell plate with 1ml of diluted Geltrex Membrane Mtrix or Matrigel Matrix at 37 ℃ for 1 h;
sixthly, digesting and centrifugally collecting cells by 0.5ml of 0.05-0.25 percent EDTA trypsin, and according to the proportion of 5 multiplied by 10 per hole4-10×104The cells are inoculated on a six-well plate coated by a matrix according to the density; adding fibroblast culture medium, 5% CO at 37 deg.C2Incubating in an incubator overnight;
seventhly, after 24 hours, replacing the fibroblast culture medium with an Essential 8 complete culture medium, and replacing the culture medium once a day; observing the appearance of induced pluripotent stem cell clones every other day under a microscope from day 8;
the colonies are usually observed to form on day 12, after 3-4 weeks the colonies grow to the appropriate size, undifferentiated stem cell colonies are manually picked, scored as first generation (P1), and transferred to petri dishes coated with Geltrex Membrane Mtrix (Gibco) for further expansion.
3. The method of constructing a bank of human induced pluripotent stem cells according to claim 2, wherein the method comprises: the fibroblast culture medium comprises the following steps: DMEM basal medium + 10% fetal bovine serum + 1% NEAA + 1% double antibody.
4. The method of constructing a bank of human induced pluripotent stem cells according to claim 1, wherein the method comprises: passage of induced pluripotent stem cells: when the Cell fusion degree reaches 80% -90%, digesting for 5-8min at 37 ℃ by using 0.5ml of digestive juice Gentle Cell Dissociation Reagent, observing the gradual Dissociation of Cell clone under a microscope, gradually loosening Cell connection, removing the digestive juice, adding 0.5ml of PBS to slightly clean residual digestive juice, removing the PBS, adding Essential 8 complete culture medium, slightly shaking the culture dish, removing the Cell clone which falls off from the bottom of the culture dish into 5-10 Cell blocks, transferring the Cell blocks into a new six-well plate, adding 2ml of Essential 8 complete culture medium, and continuing culturing.
5. The method of claim 4, wherein the method comprises the steps of: the serum-free culture medium used in the process of inducing the culture of the pluripotent stem cells is Essential 8TMMedium or StemFlexTM Medium。
6. The method of constructing a bank of human induced pluripotent stem cells according to claim 1, wherein the method comprises: cryopreservation of induced pluripotent stem cells: when the fusion degree of the passage cells reaches 80% -90%, digesting for 5-8min at 37 ℃ by using 0.5ml of Gentle Cell Dissociation Reagent, observing the gradual Dissociation of Cell clone under a microscope, gradually loosening Cell connection, removing digestive juice, adding 0.5ml of PBS to gently wash residual digestive juice, removing the PBS, adding 1ml of freezing solution, and preparing the freezing solution by KnockOutTM Serum Replacement + 10% DMSO; gently shaking the culture dish, collecting cells in a 1.8ml cryopreservation tube, putting the tube into a programmed cryopreservation box, and placing the tube in a refrigerator at the temperature of-80 ℃; after 24 hours, the cells were transferred from-80 ℃ to liquid nitrogen for storage.
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