CN103405759A - Method for preparing tumor-specific DC vaccine by applying CD34+ cells of umbilical cord blood - Google Patents
Method for preparing tumor-specific DC vaccine by applying CD34+ cells of umbilical cord blood Download PDFInfo
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Abstract
The invention discloses a method for preparing a tumor-specific DC vaccine by applying CD34+ cells in umbilical cord blood. The method comprises (1) a step of preparing autologous tumor-related holoantigen; (2) a step of obtaining the umbilical cord blood; (3) a step of obtaining mononuclear cells derived from the umbilical cord blood; (4) a step of purifying CD34+ cells in the mononuclear cells derived from the umbilical cord blood; (5) performing induction culture for a precursor DC; (6) a step of performing amplification and culture of an immature DC; and (7) a step of preparing the DC vaccine.
Description
Technical field
The present invention relates to a kind of preparation method of vaccine, particularly a kind of application umbilical blood CD34+ cell prepares tumour-specific DC vaccine method.
Background technology
The main T cellular immunization that relies on DC to induce of cellular immunization treatment of tumor.DC is the antigen presenting cell of human body sole duty, thereby, in immunotherapy of tumors, is bearing picked-up and processing tumor antigen, antigenic information is offered to the cell to T, thereby start the critical function of tumour-specific T cellular immunization.
The DC cell of clinical practice at present is mainly derived from peripheral blood list shape nucleus (Peripheral blood mononuclear cells, PBMC), and is mainly used in the treatment of individuation cellular immunization.Due to PBMC source DC(PBMC-DC) the individuation feature, limited clinical practice, particularly limited the production in enormous quantities of DC vaccine and the industrialization production of DC bacterin preparation.
Patent innovation proposition of the present invention, application Cord blood list shape nucleus (Umbilical cord blood derived mononuclear cells, UCB-MNCs), sub-elect CD34+ cell (UBC-CD34+), through 2 all amplification cultivation, be prepared into immature DC (Immature DC), 5 days load tumor antigens of amplification cultivation, ripe 48 hours (Mature DC), be prepared into tumour-specific DC vaccine.
1., umbilical blood can obtain in a large number as clinical garbage and frozen, can meet clinical practice application UBC-CD34+ cell prepared tumour-specific DC vaccine possesses following advantage:; 2., cultivate from the UBC-CD34+ cell induction, can obtain immature DC and ripe DC(UCB-CD34-DC); 3., the UBC-CD34+ cell is through the precursor DC of 14 days inducing culture, the 15-17 that quantitatively can increase doubly, is enough to meet clinical needs; 4., after the precursor DC and ripe DC cryopreservation resuscitation of UBC-CD34+ cell derived, obvious change does not occur in its cell phenotype, function, quantity; 5. with PBMC-DC, compare, the immature DC of UBC-CD34+ cell induction, antigen uptake, processing, HLA-II antigen are stronger; 6. with PBMC-DC, compare, the functional molecular on UBC-CD34-DC surface is expressed higher, and the ability of activated T cell immunity is stronger; 7. with PBMC-DC, compare, the CTL that UBC-CD34-DC activates, the tumour-specific killing ability is stronger; 8., the immunogenicity due to UBC-CD34-DC is extremely low, for allogeneic ACT treatment (Adoptive cell transfer, ACT), there are not the restriction of HLA phenotype and immunological rejection, thereby can be used for allochthonous cellular immunization treatment (Immunotherapy), broken through the individuation bottleneck of cellular immunization treatment, made DC vaccine large-scale industrialized production become possibility.
Summary of the invention
The present invention is according to prior art, and the CD34+ cell that proposes the application Cord Blood-Derived prepares the technology of DC vaccine, has obtained success, and the present invention provides a kind of preparation method of DC vaccine for this reason, described method, and step is as follows:
The preparation of step 1, the relevant holoantigen of autologous tumor:
Obtaining of step 2, umbilical blood:
Obtaining of step 3, Cord blood list shape nucleus:
The purification of step 4, Cord blood list shape nucleus CD34+ cell:
The inducing culture of step 5, precursor DC:
The amplification of step 6, immature DC and cultivation:
The preparation of step 7, DC vaccine:
Wherein each step concrete operation method is as follows:
The preparation of step 1, the relevant holoantigen of autologous tumor:
1., the fresh tumor tissues of excision: get tumor peripheral part tissue (avoiding the slough of tumor center) 0.3-1.0cm
3(but liquid nitrogen or-80 ℃ of Refrigerator stores are standby), wipe out nonneoplastic tissue on every side, gentamycin-normal saline cyclic washing 3-5 time, shred, 300 order steel meshes grind and prepare single cell suspension, and the standby tumor cell lysate of multigelation legal system is crossed the rear protein quantification of net standby.
2., the tumor cell of breast, ascites acquisition: get breast, ascites 1500-2000ml, 1200rpm is centrifugal, centrifugal 3 times of normal saline washing, and 300 order steel meshes obtain single cell suspension after crossing net, the standby tumor cell lysate of multigelation legal system, cross the rear protein quantification of net standby.
3., with organizing the derived cell strain: the multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net standby.
Get above autologous tumor lysate albumen and homologous cell strain lysate albumen each 50%, be mixed with into the relevant holoantigen of autologous tumor.
Obtaining of step 2, umbilical blood:
1., the puerpera selects: select mature or premature labor cesarean healthy women, should meet following condition: the age is below 35 years old; Without malignant tumor; Without hereditary; Without infectious diseases such as hepatitis, syphilis, AIDSs; Without serious pregnancy complications; Without family's sex-controlled inheritance disease; Without pregnancy period blood transfusion history.
2., fetus is selected: fetal weight surpasses 3000g; Without congenital diseases or deformity;
3., blood sampling contraindication: placental separation was over 12 hours; Premature rupture of fetal membrane was over 24 hours; Amniotic fluid detects finds chromosomal abnormality; During production, the puerpera has infection, heating; Fetal respiration is poverty-stricken; Meconium is arranged in amniotic fluid.
4., fetus cuts in latter 5 minutes, apart from fetus 5-7cm place dual ligation fetus side umbilical cord and in line-to-line, cuts off the 75% alcohol disinfecting broken ends of fractured bone.Capable 75% alcohol disinfecting of Placenta Hominis side umbilical cord 2-3cm, disposable blood taking device (containing anticoagulant) umbilical vein puncture extract umbilical blood (approximately 100-120ml).
5., taken a blood sample after, in 18 ℃ of storage boxes, be transported to the GMP laboratory as early as possible, and carried out cell separation in 6 hour, the latest can not be over 12 hours.
Above-mentioned umbilical blood is added to PBS dilution (0.01M by the 1:1 volume ratio, PH=7.4), 200 eye mesh screens filter, and are resuspended in the 50ml centrifuge tube that contains 15ml lymphocyte separation medium (density 1.077g/ml), 2500r/min gradient density centrifugal 20 minutes, carefully aspirate the tunica albuginea layer and obtain UCB-MNCs.
The purification of step 4, UCB-MNCs source CD34+ cell:
Beautiful day small immunomagnetic beads of the anti-CD34+ of Ni of application, by specification is from extracting the CD34+ cell of purification (after small magnetic bead sorting UCB-MNCs, cell purity is 97%, cellular morphology, phenotype, amplification ability, biological property etc. do not change, and along with passage, small magnetic bead comes off gradually, thereby does not affect cytologic experiment, and does not affect the human body application).
The inducing culture of step 5, precursor DC:
The CD34+ cell is gone respectively normal saline centrifuge washing 3 times (2000r, 1500r, 1000r/min), is inoculated in the plastic culture bottle, in containing 5% autoserous IMDM culture medium adherent 1.5 hours, removes the CD14+ cell.Collect suspension cell, according to 10
6The cell density of/ml is inoculated in culture bottle, is containing FLT3-L 25ng/ml, TPO 10ng/ml, and SCF 20ng/ml, and in 5% autoserous IMDM culture medium, 37 ℃, 5% CO
2Saturated humidity cultivated for 2 weeks, and every 2-3 days changes liquid, and 80% merges the time shift bottle, the 14th day results precursor DC(Fig. 1), can be frozen standby, can continue to induce ripe DC.
The amplification of step 6, immature DC and cultivation:
Precursor DC is inoculated in 6 orifice plates that contain 5% autoserous IMDM culture medium 2ml, and cell density is adjusted into 3 * 10
5/ well, 37 ℃, 5% CO
2In the saturated humidity incubator, adhere-wall culture is 1.5 hours, removes suspension cell, and attached cell is immature DC.In immature DC, add GM-CSF 50ng/ml, IL-4 20ng/ml, 37 ℃, 5% CO
2Saturated humidity is cultivated 5 days (half amount was changed liquid in every 2-3 days), completes the amplification cultivation (Fig. 2) of immature DC.
The preparation of step 7, DC vaccine:
Added autologous tumor be correlated with holoantigen 50 μ g/ml, TNF-α 100ng/ml, and CD40L 100ng/ml on the 5th day, and supplementary 10% autoserum, cultivated 48 hours, induce DC ripe (Fig. 3), and be prepared into tumour-specific DC vaccine, clinical vaccine therapy or frozen (cryopreservation methods: 10
6/ ml cell, add in the IMDM culture medium that contains 20% autoserum, 10%DMSO, mix, and gradient frozen plate-80 ℃ refrigerator overnight, unloading is in liquid nitrogen).
Intermediate product and end-product that above-mentioned steps of the present invention obtains detect by the following method, find that it meets the requirements fully in preparation process.
1., UCB-CD34 carrys out source precursor DC cell: the UCB-CD34+ cell induction is cultivated the precursor DC obtained in 14 days and is carried out fluidic cell detection discovery, and the precursor DC more than 90% has the CD33 high expressed, illustrates that precursor DC belongs to myelocytic series.After frozen 1 week recovery, detect, the cell sign thing does not change, and shows that cell function stablizes (table 1).
2., UCB-CD34 source immature DC antigen uptake Function detection: immature DC adds 20 μ g/ml FITC-dextran, 4 ℃ (contrast) and 37 ℃ were cultivated 30 minutes and 60 minutes altogether, PBS centrifuge washing 3 times, fluidic cell detects to be found, 30 minutes antigen uptake rates be 50%, 60 minute be 71%.Show that immature DC has powerful antigen uptake function (Fig. 4).
3., the UCB-CD34 ripe DC(DC vaccine of originating): after the immature DC amplification added antigen ripe the cultivation in 5 days, fluidic cell detects to be found, cell surface important symbol thing all has significantly and raises, and shows that the antigen presentation function of DC significantly improves (table 2).
4., mixed lymphocyte reaction (MLR) detects UCB-CD34 source DC function: get healthy human peripheral blood T cell 10
5/ well, add round bottom 96 orifice plates, by different proportion, adds ripe DC to cultivate altogether and detected discovery in 16 hours, and DC can effectively induce Allogeneic T cell proliferation (Fig. 5).
Table 1. inducing culture is in the time of 14 days, the precursor DC surface marker in UCB-CD34 source
* fresh precursor DC and each mark of frozen precursor DC surface compare, respectively P > 0.05.
The surface marker of the ripe DC in table 2. UCB-MNCs source
* each mark of fresh mature DCs and frozen mature DCs compares, respectively P > 0.05.
The UCB-CD34 DC(UCB-CD34-DC that originates) and peripheral blood list shape nucleus source DC(PBMC-DC) comparative study:
Among immaturity PBMC-DC and UBC-CD34-DCs add 20 μ g/ml FITC-dextran, 37 ℃, 5% CO 1., respectively
2Saturated humidity was cultivated respectively 30 minutes and 60 minutes.PBS centrifuge washing 3 times, fluidic cell detect to be found, the antigen uptake function of UBC-CD34-DC is higher than PBMC-DC(Fig. 6).
2., UBC-CD34-DC and PBMC-DC are respectively at cultivating the 5th day load tumor cell lysate antigen, add TNF-α to be cultured to the 7th day ELISA and detect discovery, IL-12 and INF-γ secretory volume significantly raise (P<0.01) in the PBMC-DC supernatant, in the UBC-CD34-DC supernatant, IL-6, IL-10, IL-12 and TNF-α secretory volume significantly raise (P<0.01), the IL-12 secretory volume of UBC-DC identical with PBMC-DC (Fig. 7).
3., UBC-CD34-DC and PBMC-DC load HPV-16 E7 polypeptide respectively, according to the ratio of T/DC 3:1, with CD8+T co-culture of cells (UBC-CD34-DC, A1-A3; PBMC-DC, B1-B3), the ELISApot method detects INF-γ secretory volume.Find that the ability of UBC-DC inducing T cell activation is significantly higher than inducibility (P<0.01 of PBMC-DC; Fig. 8)
4., UBC-CD34-DC and PBMC-DC induce respectively BGC-823 gastric cancer specificity; and HT-29 colon cancer specific CTL; row in vitro fragmentation test (Cytotoxicity assay) discovery, the CTL that UBC-CD34-DC induces has stronger tumour-specific lethal effect (P<0.05; Fig. 9).
The clinical practice of DC vaccine:
Vaccination ways: according to the individualized treatment principle, the DC vaccine should take drain regional lymph nodes injection system to carry out vaccine therapy.As: pulmonary carcinoma, take homonymy supraclavicular lymph nodes zone vaccine therapy; Breast carcinoma, adopt on the homonymy clavicle or axillary gland zone vaccine therapy; Rectal cancer, adopt left side inguinal lymph tie region vaccine therapy.
Clinical workflow: before DC vaccination treatment, at first applying CTX 800-1000mg disturbs tumor microenvironment, breaks tumour immunity tolerance (effectively reducing Treg, IL-10, TGF-β level), then implement the regional lymph nodes inoculation of DC vaccine, once, 3-5 time is a course for the treatment of to every 2-3 days vaccine therapy; Vein adoptive therapy (ACT) once a day or the next day once.
The accompanying drawing explanation:
Fig. 1. the precursor DC cell (* 200) in UCB-CD34 source.
Fig. 2. the amplification cultivation UCB-CD34 of the 5th day immature DC cell (* 200) of originating.
Fig. 3. the immature DC Antigen after ripening of UCB-CD34 source is cultivated and was obtained ripe DC(* 200 in 48 hours).
Fig. 4. UCB-CD34 source immature DC obviously strengthens (n=3) to the picked-up ability of FITC-dextran.
Fig. 5. mixed lymphocyte reaction (MLR) finds, the DC in UCB-MNCs source and
The DC in UCB-CD34 source, all can effectively activate normal person's periphery blood T cell.
Fig. 6. the antigen uptake function of UCB-CD34 source immature DC is higher than PBMC-DC.
Fig. 7. the IL-12 of PBMC-DC and INF-γ secretory volume obviously raise (P<0.01); The IL-6 of UCB-CD34-DC, IL-10, IL-12 and TNF-α secretory volume significantly raise (P<0.01).
Fig. 8. the ability of UCB-CD34-DC activated T cell is significantly higher than PBMC-DC(P<0.01).
Fig. 9. the CTL that UCB-CD34-DC induces, with the CTL that PBMC-DC induces, compare, have stronger tumour-specific lethal effect (P<0.05).
The specific embodiment:
Further illustrate by the following examples the present invention, but not as limitation of the present invention.
The CD34+ cell of Cord Blood-Derived prepares the preparation method of DC vaccine, and step is as follows:
The preparation of step 1, the relevant holoantigen of autologous tumor:
1., the fresh tumor tissues of excision: get tumor peripheral part tissue (avoiding the slough of tumor center) 0.3-1.0cm
3(but liquid nitrogen or-80 ℃ of Refrigerator stores are standby), wipe out nonneoplastic tissue on every side, gentamycin-normal saline cyclic washing 3-5 time, shred, 300 order steel meshes grind and prepare single cell suspension, and the standby tumor cell lysate of multigelation legal system is crossed the rear protein quantification of net standby.
2., the tumor cell of breast, ascites acquisition: get breast, ascites 1500-2000ml, 1200rpm is centrifugal, centrifugal 3 times of normal saline washing, and 300 order steel meshes obtain single cell suspension after crossing net, the standby tumor cell lysate of multigelation legal system, cross the rear protein quantification of net standby.
3., with organizing the derived cell strain: the multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net standby.
Get above autologous tumor lysate albumen and homologous cell strain lysate albumen each 50%, be mixed with into the relevant holoantigen of autologous tumor.
Obtaining of step 2, umbilical blood:
1., the puerpera selects: select mature or premature labor cesarean healthy women, should meet following condition: the age is below 35 years old; Without malignant tumor; Without hereditary; Without infectious diseases such as hepatitis, syphilis, AIDSs; Without serious pregnancy complications; Without family's sex-controlled inheritance disease; Without pregnancy period blood transfusion history.
2., fetus is selected: fetal weight surpasses 3000g; Without congenital diseases or deformity;
3., blood sampling contraindication: placental separation was over 12 hours; Premature rupture of fetal membrane was over 24 hours; Amniotic fluid detects finds chromosomal abnormality; During production, the puerpera has infection, heating; Fetal respiration is poverty-stricken; Meconium is arranged in amniotic fluid.
4., fetus cuts in latter 5 minutes, apart from fetus 5-7cm place dual ligation fetus side umbilical cord and in line-to-line, cuts off the 75% alcohol disinfecting broken ends of fractured bone.Capable 75% alcohol disinfecting of Placenta Hominis side umbilical cord 2-3cm, disposable blood taking device (containing anticoagulant) umbilical vein puncture extract umbilical blood (approximately 100-120ml).
5., taken a blood sample after, in 18 ℃ of storage boxes, be transported to the GMP laboratory as early as possible, and carried out cell separation in 6 hour, the latest can not be over 12 hours.
Above-mentioned umbilical blood is added to PBS dilution (0.01M by the 1:1 volume ratio, PH=7.4), 200 eye mesh screens filter, and are resuspended in the 50ml centrifuge tube that contains 15ml lymphocyte separation medium (density 1.077g/ml), 2500r/min gradient density centrifugal 20 minutes, carefully aspirate the tunica albuginea layer and obtain UCB-MNCs.
The purification of step 4, UCB-MNCs source CD34+ cell:
Beautiful day small immunomagnetic beads of the anti-CD34+ of Ni of application, by specification is from extracting the CD34+ cell of purification (after small magnetic bead sorting UCB-MNCs, cell purity is 97%, cellular morphology, phenotype, amplification ability, biological property etc. do not change, and along with passage, small magnetic bead comes off gradually, thereby does not affect cytologic experiment, and does not affect the human body application).
The inducing culture of step 5, precursor DC:
The CD34+ cell is gone respectively normal saline centrifuge washing 3 times (2000r, 1500r, 1000r/min), is inoculated in the plastic culture bottle, in containing 5% autoserous IMDM culture medium adherent 1.5 hours, removes the CD14+ cell.Collect suspension cell, according to 10
6The cell density of/ml is inoculated in culture bottle, is containing FLT3-L 25ng/ml, TPO 10ng/ml, and SCF 20ng/ml, and in 5% autoserous IMDM culture medium, 37 ℃, 5% CO
2Saturated humidity cultivated for 2 weeks, and every 2-3 days changes liquid, and 80% merges the time shift bottle, the 14th day results precursor DC(Fig. 1), can be frozen standby, can continue to induce ripe DC.
The amplification of step 6, immature DC and cultivation:
Precursor DC is inoculated in 6 orifice plates that contain 5% autoserous IMDM culture medium 2ml, and cell density is adjusted into 3 * 10
5/ well, 37 ℃, 5% CO
2In the saturated humidity incubator, adhere-wall culture is 1.5 hours, removes suspension cell, and attached cell is immature DC.In immature DC, add GM-CSF 50ng/ml, IL-4 20ng/ml, 37 ℃, 5% CO
2Saturated humidity is cultivated 5 days (half amount was changed liquid in every 2-3 days), completes the amplification cultivation (Fig. 2) of immature DC.
The preparation of step 7, DC vaccine:
Added autologous tumor be correlated with holoantigen 50 μ g/ml, TNF-α 100ng/ml, and CD40L 100ng/ml on the 5th day, and supplementary 10% autoserum, cultivated 48 hours, induce DC ripe (Fig. 3), and be prepared into tumour-specific DC vaccine, clinical vaccine therapy or frozen (cryopreservation methods: 10
6/ ml cell, add in the IMDM culture medium that contains 20% autoserum, 10%DMSO, mix, and gradient frozen plate-80 ℃ refrigerator overnight, unloading is in liquid nitrogen).
Claims (2)
1. an application umbilical blood CD34+ cell prepares tumour-specific DC vaccine method, described method, and step is as follows:
The preparation of step 1, the relevant holoantigen of autologous tumor:
Obtaining of step 2, umbilical blood:
Obtaining of step 3, Cord blood list shape nucleus:
The purification of step 4, Cord blood list shape nucleus CD34+ cell:
The inducing culture of step 5, precursor DC:
The amplification of step 6, immature DC and cultivation:
The preparation of step 7, DC vaccine.
2. method according to claim 1, is characterized in that, operating procedure is as follows:
The preparation of step 1, the relevant holoantigen of autologous tumor:
1., the fresh tumor tissues of excision: get the tumor peripheral part and organize 0.3-1.0cm
3, wipe out nonneoplastic tissue on every side, gentamycin-normal saline cyclic washing 3-5 time, shred, and 300 order steel meshes grind and prepare single cell suspension, and the standby tumor cell lysate of multigelation legal system is crossed the rear protein quantification of net standby;
2., the tumor cell of breast, ascites acquisition: get breast, ascites 1500-2000ml, 1200rpm is centrifugal, centrifugal 3 times of normal saline washing, and 300 order steel meshes obtain single cell suspension after crossing net, the standby tumor cell lysate of multigelation legal system, cross the rear protein quantification of net standby;
3., with organizing the derived cell strain: the multigelation legal system, for tumor cell lysate, is crossed the rear protein quantification of net standby;
Get above autologous tumor lysate albumen and homologous cell strain lysate albumen each 50%, be mixed with into the relevant holoantigen of autologous tumor;
Obtaining of step 2, umbilical blood:
, the puerpera selects: select mature or premature labor cesarean healthy women, should meet following condition: the age is below 35 years old; Without malignant tumor; Without hereditary; Without infectious diseases such as hepatitis, syphilis, AIDSs; Without serious pregnancy complications; Without family's sex-controlled inheritance disease; Without pregnancy period blood transfusion history;
2., fetus is selected: fetal weight surpasses 3000g; Without congenital diseases or deformity;
3., blood sampling contraindication: placental separation was over 12 hours; Premature rupture of fetal membrane was over 24 hours; Amniotic fluid detects finds chromosomal abnormality; During production, the puerpera has infection, heating; Fetal respiration is poverty-stricken; Meconium is arranged in amniotic fluid;
4., fetus cuts in latter 5 minutes, apart from fetus 5-7cm place dual ligation fetus side umbilical cord and in line-to-line, cut off, the 75% alcohol disinfecting broken ends of fractured bone, capable 75% alcohol disinfecting of Placenta Hominis side umbilical cord 2-3cm, the disposable blood taking device umbilical vein puncture extracts umbilical blood 100-120ml;
5., taken a blood sample after, in 18 ℃ of storage boxes, be transported to the GMP laboratory as early as possible, and carried out cell separation in 6 hour, the latest can not be over 12 hours;
Step 3, UCB-MNCs obtain:
Above-mentioned umbilical blood is added to 0.01M by the 1:1 volume ratio, the PBS dilution of PH=7.4,200 eye mesh screens filter, and are resuspended in and contain in 15ml lymphocyte separation medium 50ml centrifuge tube, and 2500r/min gradient density centrifugal 20 minutes, carefully aspirate the tunica albuginea layer and obtain UCB-MNCs;
The purification of step 4, UCB-MNCs source CD34+ cell:
Beautiful day small immunomagnetic beads of the anti-CD34+ of Ni of application, by specification is from extracting the CD34+ cell of purification UCB-MNCs;
The inducing culture of step 5, precursor DC:
The CD34+ cell is gone respectively normal saline centrifuge washing 3 times, is inoculated in the plastic culture bottle, and in containing 5% autoserous IMDM culture medium adherent 1.5 hours, remove the CD14+ cell, collect suspension cell, according to 10
6The cell density of/ml is inoculated in culture bottle, is containing FLT3-L 25ng/ml, TPO 10ng/ml, and SCF 20ng/ml, and in 5% autoserous IMDM culture medium, 37 ℃, 5% CO
2Saturated humidity cultivated for 2 weeks, and every 2-3 days changes liquid, and 80% merges the time shift bottle, and the 14th day results precursor DC, can be frozen standby, can continue to induce ripe DC;
The amplification of step 6, immature DC and cultivation:
Precursor DC is inoculated in 6 orifice plates that contain 5% autoserous IMDM culture medium 2ml, and cell density is adjusted into 3 * 10
5/ well, 37 ℃, 5% CO
2In the saturated humidity incubator, adhere-wall culture is 1.5 hours, removes suspension cell, and attached cell is immature DC, in immature DC, adds GM-CSF 50ng/ml, IL-4 20ng/ml, 37 ℃, 5% CO
2Saturated humidity was cultivated 5 days, completed the amplification cultivation of immature DC;
The preparation of step 7, DC vaccine:
Added autologous tumor be correlated with holoantigen 50 μ g/ml, TNF-α 100ng/ml, and CD40L 100ng/ml on the 5th day, and supplement 10% autoserum, cultivated 48 hours, induce the DC maturation, and be prepared into tumour-specific DC vaccine, clinical vaccine therapy or frozen.
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| CN106434557A (en) * | 2016-11-25 | 2017-02-22 | 博雅干细胞科技有限公司 | Method for preparing CD34 positive cells from umbilical cord mesenchymal stem cells |
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