CN113331176A - Mesenchymal stem cell cryopreservation liquid - Google Patents
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Abstract
The invention relates to a mesenchymal stem cell frozen stock solution which comprises a Caspase monoclonal antibody, wherein the monoclonal antibody has better capability of combining Caspase protein. After the corresponding antibody is added into the frozen stock solution, the survival rate of the cells after frozen stock can be effectively improved, the preservation and the application of the umbilical cord mesenchymal stem cells are facilitated, and the application value is good.
Description
Technical Field
The invention relates to the field of biology, and particularly relates to a mesenchymal stem cell cryopreservation solution.
Background
The umbilical cord blood contains relatively rich hematopoietic stem cells and mesenchymal stem cells, and the mesenchymal stem cells can be induced and differentiated into various histiocytes in vitro under certain conditions, so that the method is beneficial to repairing diseased or damaged histiocytes, is widely applied to clinic, and is mainly applied to repairing and regenerating malignant tumors, cartilages and bones, repairing damaged nerves, treating liver diseases and the like in the blood system at present.
In recent years, cord blood stem cell transplantation has become one of the important means for clinical treatment of various malignant and non-malignant hematological diseases. Previous researches suggest that a fusion gene of a SCL gene and an TALL gene of acute T-lymphocyte leukemia plays an important role in a hematopoietic system, scholars transfect the SCL and the TALL genes into cord blood CD34+ stem cells through viruses, and experiments show that the SCL, the TALL gene and MEK/ERK phosphorylation in the cord blood CD34+ stem cells have influence on the hematopoietic function of organisms, and the SCL and the TALL genes play a dominant role in the stem cell and can regulate the MEK/ERK phosphorylation of a signal pathway. Meanwhile, because the umbilical cord blood contains a large amount of mesenchymal stem cells, the umbilical cord blood can promote the proliferation and differentiation of hematopoietic stem cells through the effects of intercellular action, secretion of hematopoietic growth factors and cytokines and the like. The experimental result shows that the mesenchymal stem cells transplanted by combining the umbilical cord blood are an effective treatment method, and the mesenchymal stem cells can reduce the occurrence of graft rejection and the recurrence probability of blood tumor. Also, researchers have found that the proliferation capacity of lymphocytes is inhibited after combined culture by combining umbilical cord blood mesenchymal stem cells and lymphocytes, particularly CD8+ lymphocyte subtypes, but CD4+ T lymphocyte subtypes are significantly increased, and in addition, umbilical cord blood mesenchymal stem cells can reduce Thl and Tcl cell populations and increase Th2 and Te2 cell populations, so that umbilical cord blood stem cells are considered to have greater potential in inhibiting and reducing clinical graft-versus-host disease. In addition, the UCBT has the advantages of rich sources, low immunogenicity, potential of differentiation into various hematopoietic cells and the like, can promote the homing of HSCs by releasing various chemotactic factors, cytokines and adhesion factors, has obvious promotion effects on promoting the maturation of a hematopoietic microenvironment of an organism, accelerating the reconstruction of a hematopoietic system and the like, and becomes one of the hot spots of the current research on the diseases of the blood system.
Although cord blood stem cells have the advantages of abundant sources, convenient collection, no harm to donors, no limitation by ethics and laws, stronger differentiation capability, lower immunogenicity and the like, so that the cord blood stem cells show considerable clinical prospects in the aspect of treatment of the diseases, the cord blood stem cells have many problems before the clinical technology is mature. The low absolute value of stem cells in the umbilical cord blood limits the clinical application of the stem cells, so how to improve the culture method and the culture success rate, particularly the method of cryopreservation, is an urgent problem to be solved.
Previous researches show that with deep understanding of the low-temperature cryopreservation theory, people find that cell apoptosis participates in a molecular mechanism of cell freezing damage, and the hematopoietic stem cell apoptosis seriously damages the cloning forming capability and the hematopoietic reconstruction function after transplantation. Caspase family members are important effector proteases of apoptosis cascade reactions, and the survival rate of various cells after cryopreservation can be improved by inhibiting the expression of Caspase. The Caspase inhibitor zVAD-fmk is used for cryopreserving the stem cells, and a better cryopreserving effect is found. However, in the current research, the variety of Caspase inhibitors is not abundant enough, the number of alternative types is not enough, and the influence of zVAD-fmk on the cell is not completely inactive, so that the research of Caspase inhibitors is urgent.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a caspase monoclonal antibody and application thereof.
In one aspect, the invention provides a caspase monoclonal antibody, wherein the sequences of a light chain variable region and a heavy chain variable region of the monoclonal antibody are respectively shown as follows:
light chain variable region (SEQ ID NO: 1)
DIVITQSPALMAASPGEKVTITCCPMKSAACICMKWYQQKSGISPKPWIYEYHYDTQGVPARFSGSGSGTSYSLTITSMEAEDAATYYCHYVEVDAGSFGAGTKLELK
Heavy chain variable region (SEQ ID NO: 2)
EVQLEESGTELARPGASVKLSCKASGYIFSCVHQWWIKQRPGQGLEWIGKGQHDMCGNRGTVNPYGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGDNEAQGNWGLGTTLAVSS。
Further, the present invention provides an umbilical cord mesenchymal stem cell, the preparation method of which comprises: taking 15mL of umbilical cord blood, performing heparin anticoagulation, adding an equal amount of phosphate buffer solution for dilution, taking 50mL of a centrifuge tube, firstly adding 15mL of Percoll gradient separation solution with the density of 1.077g/mL, carefully adding the diluted umbilical cord blood, centrifuging at 2000r/min for 25min, sucking white annular floccules (rich in mononuclear cells) of a layered interface by using a pipette, centrifuging and washing at 800r/min for 2 times and 5 min/time, suspending deposited cells by using a DMEM high-sugar medium containing 15% fetal calf serum, 100U/mL penicillin and 100U/mL streptomycin, adjusting the cell density to be L multiplied by 109/L, inoculating the cells into a 25T culture bottle, placing the culture bottle in a C0 culture bottle with the temperature of 37 ℃ and the volume fraction of 0.052Culturing in a saturated humidity incubator. After incubation for 72h, the medium was changed, erythrocytes and other non-adherent cells were removed, and the solution was changed 1 time for three days. The mesenchymal stem cells are mutually confluent at about 7 weeks to reach 70-80% of the growth surface of the bottom of the culture bottle, and at the moment, subculture is carried out. Pouring out old culture medium, adding 2mL of 0.25% pancreatin for digestion, removing most part after 30s, and reserving a little in bottle, and adaptingAnd (3) when the culture bottle is shaken vigorously, the whole bottle of cells is placed in the culture box for 5min, the dissociation degree of the cells is observed under an inverted microscope, the DMEM high-sugar medium containing 15% fetal calf serum is added to the cells with the cell processes of retraction, cell gap enlargement and approximate cell roundness as degrees, the digestion is stopped, the bottom of the bottle is blown by a suction pipe gently, the bottle is washed by centrifugation at 800r/min for 2 times and 5 min/time, and the collected cells are the umbilical cord mesenchymal stem cells of the passage.
Further, the invention provides a cryopreservation protective solution for umbilical cord mesenchymal stem cells, which is prepared by mixing an IMDM culture medium, fetal bovine serum and dimethyl sulfoxide according to a volume ratio of 7:2:1, wherein 30 mu mol/L of monoclonal antibody is added.
Further, the method of using the cryopreservation solution of the present invention is to adjust the cell density to 1 × 10 in the cryopreservation protection solution5And/ml, storing in liquid nitrogen according to the following program of 15min at 4 deg.C, 2h at-20 deg.C, overnight at-80 deg.C, and freezing at-196 deg.C. After the cells were frozen at-196 ℃ for 4 weeks, they were immediately placed in a 40 ℃ water bath and thawed within 2min, and then washed with IMDM medium to which the monoclonal antibody of example 1 (30. mu. mol/L) was added.
The expression inhibition of caspase protein in the cells after freezing storage can be detected, and the inhibition rate of the expression amount of the protein is more than 97% compared with the cells after freezing storage without adding the antibody. The antibody can improve the revival rate after cryopreservation, and the cell revival rate of the cryopreservation solution can still reach over 90 percent after the cryopreservation for 2 years.
Furthermore, other common freezing preservation protective agents can be added into the freezing preservation liquid.
Advantageous effects
The Caspase monoclonal antibody provided by the invention has good capability of combining Caspase protein, can effectively improve the survival rate of cells after cryopreservation by adding the corresponding antibody into the cryopreservation solution, is beneficial to the storage and application of umbilical cord mesenchymal stem cells, and has good application value.
Drawings
FIG. 1 Sox9 mRNA relative expression level results
FIG. 2 Sox9 protein relative expression level results
Detailed Description
In order to make the aforementioned and other objects, features and advantages of the invention more comprehensible, preferred embodiments accompanied with figures are described in detail below.
EXAMPLE 1 preparation of Caspase mAb
Animal immunization: purified recombinant human Caspase-3 protein 5mg (BioVision, cat # 1083-5) was mixed well with equal volume of Freund's complete adjuvant and BALB/c mice were injected subcutaneously with 50. mu.g/mouse of fusion protein at multiple points. The primary immunization was performed at 4 weeks, and the postero-cutaneous multi-point injection was performed at the back, and the secondary immunization was performed at 4 weeks, with 50. mu.g of immunogen used per time. And (4) after 3 rd immunization, measuring the serum antibody titer by ELISA (enzyme-linked immunosorbent assay) in tail vein blood collection for 8-10 d. The 1 mouse with the highest antibody titer was selected 4d before the fusion and boosted by intraperitoneal injection of 150. mu.g of non-adjuvant antigen. Cell fusion and screening: taking splenocytes of the boosting BALB/c mice aseptically, taking 50% polyethylene glycol as a fusion agent, and mixing the mixture according to the proportion of 1: 5-1: 10 fusing Sp2/0 myeloma cells with mouse splenocytes, adding HAT selective culture solution, inoculating a 96-well plate, culturing in an incubator at 37 ℃ and 5% CO2, sucking culture supernatant in growth cloning wells after 7-10 days, screening by ELISA (enzyme-linked immunosorbent assay) by using target protein as coating antigen, screening 18 strains of positive hybridoma only reacting with the target protein, and carrying out 4 rounds of subcloning on 2 strains of hybridoma with the strongest positive reaction by adopting a limiting dilution method until all the monoclones are positive, and carrying out expanded culture and strain establishment.
Preparation and purification of monoclonal antibody: shaking BALB/c mouse intraperitoneal injection hybridoma cell 1X106And after about 7 days, after the abdomen of the mouse expands remarkably, sucking ascites at 2000r/min, centrifuging for 20min, and taking the supernatant. After dilution with equilibration buffer, the mixture was filtered through a 0.45 μm filter. Passing the filtered ascites through a Purify protein purifier at a flow rate of 1ml/min, eluting with a balanced buffer solution to remove impure proteins, eluting with an eluent (glycine-hydrochloric acid pH2.8100mmol/L) to elute antibody, and collecting OD280>0.5 antibody, which was subjected to SDS-PAGE electrophoresis after dialysis with PBS 4.
Detecting monoclonal antibody Westernblot: the immune proteins were added to 2 xSDS lysates, respectively, and loaded at 5ng per lane. After SDS-PAGE, proteins were transferred to nitrocellulose membranes; after 5% skimmed milk powder (prepared in TBST) was sealed for 2h at room temperature, the cells were incubated with 4 g of monoclonal antibody (2. mu.g/ml) overnight. After the next day of washing, HRP-labeled goat anti-mouse IgG (1: 2000) was added, incubated at room temperature for 50min, and ECL chemiluminescence was developed.
In the Westernblot experiment, the result shows that the monoclonal antibody can be specifically combined with the corresponding immune protein and not combined with other proteins.
The sequences of the light chain variable region and the heavy chain variable region of the monoclonal antibody obtained by a conventional antibody sequence identification method are respectively shown as follows: light chain variable region (SEQ ID NO: 1)
DIVITQSPALMAASPGEKVTITCCPMKSAACICMKWYQQKSGISPKPWIYEYHYDTQGVPARFSGSGSGTSYSLTITSMEAEDAATYYCHYVEVDAGSFGAGTKLELK
Heavy chain variable region (SEQ ID NO: 2)
EVQLEESGTELARPGASVKLSCKASGYIFSCVHQWWIKQRPGQGLEWIGKGQHDMCGNRGTVNPYGKATLTADKSSSTAYMQLSSLASEDSAVYYCAGDNEAQGNWGLGTTLAVSS。
The affinity of the antibody and the immune protein is 2.8nM by using a conventional affinity identification method.
Example 2 preparation of umbilical cord mesenchymal stem cells
Separating and culturing umbilical cord blood mesenchymal thousand cells: taking 15mL of umbilical cord blood, performing heparin anticoagulation, adding an equal amount of phosphate buffer solution for dilution, taking 50mL of a centrifuge tube, firstly adding 15mL of Percoll gradient separation solution with the density of 1.077g/mL, carefully adding the diluted umbilical cord blood, centrifuging at 2000r/min for 25min, sucking white annular floccules (rich in mononuclear cells) of a layered interface by using a pipette, centrifuging and washing at 800r/min for 2 times and 5 min/time, suspending deposited cells by using a DMEM high-sugar medium containing 15% fetal calf serum, 100U/mL penicillin and 100U/mL streptomycin, adjusting the cell density to be L multiplied by 109/L, inoculating the cells into a 25T culture bottle, placing the culture bottle in a C0 culture bottle with the temperature of 37 ℃ and the volume fraction of 0.052Culturing in a saturated humidity incubator. After incubation for 72h, the medium was changed, erythrocytes and other non-adherent cells were removed, and the solution was changed 1 time for three days. The mesenchymal stem cells are mutually confluent at about 7 weeks to reach 70-80% of the growth surface of the bottom of the culture bottle, and at the moment, subculture is carried out. Pouring out the old culture medium, adding 2mL of 0.25% pancreatin for digestion, removing most of the culture medium after 30s of action,and (3) reserving a little of the mixture in a bottle, shaking the culture bottle with proper force, putting the whole bottle of cells into an incubator for 5min, observing the dissociation degree of the cells under an inverted microscope, adding a DMEM high-sugar culture medium containing 15% fetal calf serum to stop digestion according to the degrees that the cell protrusions retract, the cell gaps increase and the cells are approximately round, blowing and beating the bottom of the bottle by a suction pipe softly, centrifugally washing for 2 times at 800r/min for 5 min/time, and collecting the cells, namely the passage umbilical cord mesenchymal stem cells.
And (3) detecting the immunological phenotype of the umbilical cord mesenchymal stem cells by an immunofluorescence method. Conventional umbilical cord mesenchymal stem cell climbing films are washed by 0.1M PBS for 5min3 times, fixed by 4% paraformaldehyde for 30min, washed by 0.1M PBS for 5min3 times, serum is sealed for 30min, primary anti-CD 44 and CD105(1:100) are dripped for overnight at 37 ℃ for 1h or 4 ℃, washed by 0.1M PBS for 5min3 times, FITC labeled secondary antibodies are dripped, incubated at room temperature in dark for 30min, washed by 0.1M PBS for 30min, PE is used for nuclear staining, and observation and photography are carried out under a fluorescence microscope. As a result, the result shows that the umbilical cord mesenchymal stem cells are observed under a fluorescence microscope and show CD 44-FITC and CD 105-FITC positivity, which indicates that umbilical cord mesenchymal stem cells are obtained by separation and passage.
Example 2 cryopreservation experiment of umbilical cord mesenchymal stem cells
The cells were divided into three groups, and the following 3 different compositions of cryopreservation solutions were added (the formulations in each group were volume ratio).
Freezing medium for group A (mixed by IMDM medium, fetal calf serum and dimethyl sulfoxide at ratio of 7:2: 1)
Adjusting cell density to 1x105And/ml, storing in liquid nitrogen according to the following program of 15min at 4 deg.C, 2h at-20 deg.C, overnight at-80 deg.C, and freezing at-196 deg.C. After the cells were frozen at-196 ℃ for 4 weeks, they were immediately placed in a 40 ℃ water bath, thawed within 2min, and washed with IMDM medium for further use (conventional frozen group).
B frozen stock solution (prepared by mixing IMDM culture medium, fetal bovine serum and dimethyl sulfoxide at a ratio of 7:2:1, adding zVAQ-fmk (30. mu. mol/L), and adjusting cell density to 1x105And/ml, storing in liquid nitrogen according to the following program of 15min at 4 deg.C, 2h at-20 deg.C, overnight at-80 deg.C, and freezing at-196 deg.C. Freezing the cells at-196 deg.C for 4 weeks, immediately placing into 40 deg.C water bath box, and thawing within 2minThe cells were washed with IMDM medium (to which zVAQ-fmk (30. mu. mol/L) was added) until use.
Group C frozen stock solution (prepared by mixing IMDM medium, fetal bovine serum and dimethyl sulfoxide at a ratio of 7:2:1, adding monoclonal antibody (30. mu. mol/L) of example 1, and adjusting cell density to 1X105And/ml, storing in liquid nitrogen according to the following program of 15min at 4 deg.C, 2h at-20 deg.C, overnight at-80 deg.C, and freezing at-196 deg.C. After the cells were frozen at-196 ℃ for 4 weeks, they were immediately placed in a 40 ℃ water bath and thawed within 2min, and then washed with IMDM medium to which the monoclonal antibody of example 1 (30. mu. mol/L) was added.
Adjusting the number of cells to 5X10 before cryopreservation5and/mL, after trypan blue staining, randomly selecting 6 visual fields in each group under a common light microscope, counting the number of cells stained by trypan blue, and calculating the survival rate of the cells as [ (total number of cells-number of trypan blue stained cells)/the total number of counted cells X100%. The results are shown in table 1 below.
TABLE 1 Effect of different groups of frozen stock solutions on the survival rate of resuscitated cells
| Group of | The survival rate of the cells is% |
| A | 80.1±2.3 |
| B | 93.1±3.3 |
| C | 95.7±2.2 |
As can be seen from Table 1, the monoclonal antibodies of the present invention have similar effects on protecting cell cryopreserving as zVAQ-fmk capsase inhibitors.
Example 3 induced differentiation experiment of cells after cryopreservation
Induced differentiation of umbilical cord blood mesenchymal stem cells into chondrocytes: taking three groups of revived cells after freezing storage according to the density of 5X107L-1Inoculating into 24-well culture plate (sterile cover glass placed in the well), changing the culture solution on day 2, culturing half of culture wells with culture medium for inducing chondrocyte (DMEM containing 15% fetal bovine serum and insulin-like growth factor 1 with final concentration of 100 pg/L), changing the culture solution 1 time after 3d, and inducing for 12 d.
Collecting cell mass of cartilage induction 12d, removing culture medium by aspiration, washing with PBS for 2 times, adding appropriate amount of RNAasso plus, and repeatedly blowing to fully crack cells. Extracting total RNA and reverse transcription to synthesize cDNA. And (3) performing real-time PCR by using the cDNA as a template and using a SYBR Green MasterMix reagent to detect the expression condition of the target gene. The primer sequences used are shown below.
Beta-actin primer sequence (5 '→ 3')
F：AGATGTGATCAGCAAGCAG
R：GCGCAAGTTAGGTTTTGTCA
Primer sequence of hSox9 (5 '→ 3')
F：CACGGAGCAGACGCACATC
R: GGCTGCACGTCGGTTTTGG, the results are shown in FIG. 1.
During the induced differentiation process, compared with an uninduced group, the expression of the cartilage differentiation marker gene Sox9 at the mRNA level is obviously increased after 12d induction. Particularly, the mRNA expression level after the antibody is added into the induction group and the freezing treatment is improved to 34.6, and the effect is obvious.
Respectively collecting cell clusters integrated with cartilage induction for 12d, absorbing and removing the culture medium, lightly washing with PBS for 2 times, adding a proper amount of protein lysate containing protein inhibitor, and repeatedly freezing and thawing to fully lyse the cells. Protein concentration was determined using the BCA protein concentration detection kit according to the instructions. 30 μ g of protein per well was separated by 12% SDS-PAGE gel electrophoresis, and then transferred to PVDF membrane by semidry method. Blocking with 5% skimmed milk powder solution prepared from TBST for 2h, adding corresponding primary antibody, and incubating overnight at 4 deg.C. And (3) incubating for 2h at room temperature by using a horseradish peroxidase (HRP) labeled secondary antibody, finally, uniformly dripping ECL luminescent solution on the membrane, incubating for 1min at room temperature, and then, exposing in a gel imager to photograph.
Western blot detection of the expression of cartilage differentiation marker gene Sox9 shows that the expression of Sox9 protein level in the frozen stock solution treatment group containing antibody treatment is obviously increased, and the expression of group C is up to 3.54. The results show that the stem cells treated by the monoclonal antibody have better cell activity maintenance, can better promote chondrogenic differentiation of the stem cells and improve the chondrogenic differentiation capacity.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
<110> Beijing Biotechnology Ltd
<120> mesenchymal stem cell cryopreservation liquid
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Claims (5)
1. A caspase monoclonal antibody, wherein the variable region sequence of the light chain of the monoclonal antibody is shown as SEQ ID NO: 1, the sequence of the heavy chain variable region is shown as SEQ ID NO: 2, respectively.
2. The application of the caspase monoclonal antibody in preparing umbilical cord blood mesenchymal stem cell frozen stock solution according to claim 1; wherein the frozen stock solution is prepared by mixing IMDM culture medium, fetal bovine serum and dimethyl sulfoxide according to the volume ratio of 7:2:1, and the monoclonal antibody in the claim 1 is added to the frozen stock solution until the final concentration is 30 mu mol/L.
3. An umbilical cord blood mesenchymal stem cell frozen stock solution, wherein the frozen stock solution is prepared by mixing an IMDM culture medium, fetal bovine serum and dimethyl sulfoxide according to a volume ratio of 7:2:1, and the monoclonal antibody in the claim 1 is further added into the frozen stock solution to a final concentration of 30 μmol/L.
4. The use of the cryopreservation solution of claim 3 in cryopreservation of umbilical cord blood mesenchymal stem cells; the application method of the frozen stock solution comprises adjusting cell density to 1x10 in the frozen stock protection solution5And/ml, storing in liquid nitrogen according to the following program, namely storing at 4 ℃ for 15min, at-20 ℃ for 2h, at-80 ℃ overnight, finally freezing at-196 ℃, immediately placing the cells in a water bath at 40 ℃ after freezing at-196 ℃ for 4 weeks, completely thawing within 2min, and washing with IMDM culture medium to which 30 mu mol/L of the monoclonal antibody of claim 1 is added for later use.
5. The cryopreservation solution of claim 3, wherein other conventional cryopreservation protecting agents can be added.
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| CN114042030A (en) * | 2021-11-29 | 2022-02-15 | 北京戴域生物技术有限公司 | Cosmetic and anti-inflammatory drug containing adipose-derived mesenchymal stem cell freeze-dried powder |
| CN116270413A (en) * | 2023-04-19 | 2023-06-23 | 广州研智化妆品有限公司 | Preparation method of cosmetic containing adipose-derived mesenchymal stem cell freeze-dried powder |
| CN116889228A (en) * | 2023-07-12 | 2023-10-17 | 重庆市铂而斐细胞生物技术有限公司 | Cryopreservation method of umbilical cord mesenchymal stem cells |
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