CN116426464A - Culture medium combination, preparation method and application of mesenchymal stem cells - Google Patents

Culture medium combination, preparation method and application of mesenchymal stem cells Download PDF

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CN116426464A
CN116426464A CN202211536629.9A CN202211536629A CN116426464A CN 116426464 A CN116426464 A CN 116426464A CN 202211536629 A CN202211536629 A CN 202211536629A CN 116426464 A CN116426464 A CN 116426464A
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culture medium
concentration
medium
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stem cells
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郭昌龙
马旭
董艺超
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Institute Of Science And Technology National Health Commission
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Abstract

The present invention provides a culture medium combination that can induce differentiation of Pluripotent Stem Cells (PSC) into Mesenchymal Stem Cells (MSC). The invention also provides a preparation method of the PSC-derived MSC, which has the characteristics of short period, high efficiency, simple operation, infinite expansion and the like, and the obtained MSC accords with the identification standard, is safe and stable, does not have the tumorigenicity, has the function similar to that of the umbilical cord-derived MSC, and can be used for the treatment of wound repair.

Description

Culture medium combination, preparation method and application of mesenchymal stem cells
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a culture medium combination, a preparation method of mesenchymal stem cells from a multipotent stem cell differentiation source and application thereof.
Background
Pluripotent stem cells (pluripotent stem cell, PSC), including embryonic stem cells (embryonic stem cell, ESC) and induced pluripotent stem cells (inducedpluripotent stem cell, iPSC), are a class of cell populations with self-renewing and multipotent differentiation potential.
Mesenchymal stem cells (MSC, mesenchymal stem cells) are an important member of the stem cell family, derived from the early stages of development mesoderm, originally found in bone marrow. The stem cells have the characteristics of multidirectional differentiation potential, hematopoietic support, stem cell implantation promotion, immune regulation, self replication and the like, and are paid attention to. For example, the mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and the like under specific induction conditions in vivo or in vitro, and still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue organ injury caused by aging and pathological changes. The clinical application of the mesenchymal stem cells in solving various blood system diseases, cardiovascular diseases, liver cirrhosis, nervous system diseases, knee joint meniscus partial excision injury repair, autoimmune diseases and the like is a major breakthrough, and the lives of more patients are saved. In addition, mesenchymal stem cells have long-term development prospects in other aspects such as regenerative medicine.
Although MSCs currently have great therapeutic potential in regenerative medicine, due to the complex tissue sources of MSCs and the lack of agreement of in vitro expansion systems, MSCs of adult origin have strong donor dependence and batch-to-batch heterogeneity. First, MSCs of different tissue sources have a great difference in functional properties, as found by research, BM-MSCs have a stronger osteogenic differentiation ability, whereas MSCs of umbilical cord, placenta and fat source have a weaker osteogenic differentiation ability. Secondly, even MSCs of single tissue origin have strong donor dependence, and even the isolated primary cells are difficult to achieve complete uniformity in cell quality level under the interference of unstable factors such as donor age, physical condition difference, and the like. In addition, in the isolated culture process of the cells, the replicative aging problem of the cells in the culture process is aggravated due to the imperfect culture conditions, so that the immunophenotype and differentiation capacity of the cells in the higher generation are changed, and the functional characteristics of the cells are far less than those of the primary cells. Although MSCs have great therapeutic potential in regenerative medicine, they are not well applied to clinic at this stage due to the limitations of the above factors.
Therefore, how to solve the problems of complex MSC sources, insufficient extractable quantity and non-uniform batch-to-batch quality becomes a key for advancing MSC application to the next stage.
Disclosure of Invention
In some embodiments, the invention provides a combination of media.
In some embodiments, the medium combination comprises medium a, medium B, medium C, medium D, and medium E.
In some embodiments, the medium a is a medium to which BMP4 and activin a are added to the complete medium.
In some embodiments, the medium B is a medium with BMP4 added to the complete medium.
In some embodiments, the medium C is a medium to which ALK inhibitors are added in complete medium.
In some embodiments, the medium D is a medium to which bFGF is added in complete medium.
In some embodiments, the medium E is a medium in which FBS, VEGF, EGF, IGF-1, TGF-beta, and bFGF are added to a basal medium.
In some embodiments, the ALK inhibitor comprises SB431542.
In some embodiments, the complete medium comprises E6 medium and E8 medium.
In some embodiments, the complete medium is E6 medium.
In some embodiments, the basal medium comprises DMEM/F12, DMEM, a-MEM and knock out DMEM/F12.
In some embodiments, the basal medium is DMEM/F12.
In some embodiments, the BMP4 concentration in medium A is 0.5-50 μg/L, activin A concentration is 0.5-30 μg/L, BMP4 concentration in medium B is 0.5-30 μg/L, SB431542 concentration in medium C is 0.5-10 μg/L, bFGF concentration in medium D is 0.5-10 μg/L, FBS concentration in medium E is 2-15%, VEGF concentration is 0.5-10 μg/L, EGF concentration is 0.5-10 μg/L, IGF-1 concentration is 0.5-10 μg/L, TGF-B concentration is 0.5-10 μg/L, bFGF concentration is 0.5-10 μg/L.
In some embodiments, the BMP4 concentration in medium A is 5-25 μg/L, the activin A concentration is 5-25 μg/L, the BMP4 concentration in medium B is 5-25 μg/L, the SB431542 concentration in medium C is 1-10 μg/L, the bFGF concentration in medium D is 1-10 μg/L, the FBS concentration in medium E is 2-10%, the VEGF concentration is 1-10 μg/L, EGF concentration is 1-10 μg/L, IGF-1 concentration is 1-10 μg/L, TGF-B concentration is 1-10 μg/L, bFGF concentration is 1-10 μg/L.
In some embodiments, the BMP4 concentration in medium A is 5-20 μg/L, the activin A concentration is 5-20 μg/L, the BMP4 concentration in medium B is 5-20 μg/L, the SB431542 concentration in medium C is 2-8 μg/L, the bFGF concentration in medium D is 2-8 μg/L, the FBS concentration in medium E is 2-8%, the VEGF concentration is 1-8 μg/L, EGF concentration is 1-8 μg/L, IGF-1 concentration is 1-8 μg/L, TGF-B concentration is 1-8 μg/L, bFGF concentration is 1-8 μg/L.
In some embodiments, the BMP4 concentration in medium A is 5-15 μg/L, the activin A concentration is 5-15 μg/L, the BMP4 concentration in medium B is 5-15 μg/L, the SB431542 concentration in medium C is 4-6 μg/L, the bFGF concentration in medium D is 4-6 μg/L, the FBS concentration in medium E is 4-6%, the VEGF concentration is 1-5 μg/L, EGF concentration is 1-5 μg/L, IGF-1 concentration is 1-5 μg/L, TGF-B concentration is 1-5 μg/L, bFGF concentration is 1-5 μg/L;
in some embodiments, the BMP4 concentration in medium A is 10 μg/L and the activin A concentration is 10 μg/L.
In some embodiments, the BMP4 concentration in medium B is 10 μg/L.
In some embodiments, the concentration of SB431542 in medium C is 5uM.
In some embodiments, the bFGF concentration in medium D is 5. Mu.g/L.
In some embodiments, the medium E has a FBS concentration of 5%, a VEGF concentration of 2 μg/L, EGF, a 2 μg/L, IGF-1, a 2 μg/L, TGF-b concentration, a 2 μg/L, bFGF concentration, and a 2 μg/L concentration.
In some embodiments, the medium combination further comprises medium F.
In some embodiments, the medium F is a medium to which FBS, VEGF, EGF, IGF-1, TGF-b, bFGF, p38MAPK inhibitor is added to the basal medium.
In some embodiments, the p38MAPK inhibitor is SB202190.
In some embodiments, medium F has a FBS concentration of 5%, VEGF concentration of 2. Mu.g/L, EGF concentration of 2. Mu.g/L, IGF-1 concentration of 2. Mu.g/L, TGF-b concentration of 2. Mu.g/L, bFGF concentration of 2. Mu.g/L, SB202190 concentration of 5. Mu.M.
In some embodiments, the basal medium comprises DMEM/F12, DMEM, a-MEM, knock out DMEM/F12.
In some embodiments, the basal medium is DMEM/F12.
In some embodiments, the present invention also provides a method for preparing mesenchymal stem cells from multipotent stem cell differentiation, comprising the steps of:
(1) Culturing pluripotent stem cells to be induced;
(2) Sucking and removing the culture medium of the pluripotent stem cells to be induced in the step (1), adding the culture medium A, and continuing to culture;
(3) Completely sucking the culture medium A, adding the culture medium B, and continuing to culture;
(4) Completely sucking and removing the culture medium B, adding the culture medium C, and continuing to culture;
(5) Completely sucking and removing the culture medium C, adding the culture medium D, and continuing to culture;
(6) Sucking half of the volume of the culture medium D, adding half of the volume of the culture medium E, and continuing to culture;
(7) Completely sucking and removing the culture medium in the step (6), adding a fresh culture medium E, and continuously culturing to obtain the mesenchymal stem cells.
In some embodiments, the pluripotent stem cells comprise embryonic stem cells and induced pluripotent stem cells.
In some embodiments, the pluripotent stem cells are induced pluripotent stem cells.
In some embodiments, in step (1), the medium of the pluripotent stem cells is selected from one of mTESR1 medium, teSR-E8, stemflex medium.
In some embodiments, in step (1), the culture medium of the pluripotent stem cells is TeSR-E8 culture medium.
In some embodiments, the method further comprises passaging the mesenchymal stem cells obtained in step (6).
In some embodiments, the passaging comprises inoculating the cells into medium F for culturing.
In some embodiments, the present invention also provides mesenchymal stem cells prepared by the above method.
In some embodiments, the mesenchymal stem cells highly express CD73, CD90, CD105, while lowing CD14, CD34, CD45, CD79a, and HLA-DR.
In some embodiments, the invention provides the use of the mesenchymal stem cells described above for the preparation of a medicament for treating a disease.
In some embodiments, the disease comprises wound repair.
In some embodiments, the wound is a skin lesion.
In some embodiments, the skin injury is a wound of a diabetic patient.
The culture medium combination provided by the invention can induce and differentiate pluripotent stem cells (pluripotent stem cell, PSC) into Mesenchymal Stem Cells (MSC), has the characteristics of short period, high efficiency, simplicity in operation, infinite expansion and the like, can obtain MSC meeting identification standards, is safe and stable, does not have tumorigenicity, has functions similar to umbilical cord-derived MSC, and can be used for wound repair treatment. Compared with the MSC from the traditional tissue, the mesenchymal stem cells prepared by the invention can effectively solve the problem of insufficient quantity of primary tissue cells, radically solve the problem of donor dependence and batch-to-batch heterogeneity of the mesenchymal stem cells, and provide powerful guarantee for the application of the mesenchymal stem cells in regenerative medicine.
Drawings
Fig. 1 is a microscopic view of morphological changes of iPSC differentiation into iMSC.
Fig. 2 is a microscopic view of hmscs of generation P0, P6 and P10.
FIG. 3 shows the results of surface molecular flow identification of iMSC.
Fig. 4 shows the results of subcutaneous tumorigenesis experiments with imscs.
FIG. 5 shows the results of the test for the tumorigenicity of A549 cells and iMSC.
FIG. 6 shows the results of in vivo metabolism detection of iMSC.
FIG. 7 shows the expression of multipotent factors in iMSC.
FIG. 8 shows the results of the telomerase activity assay of iMSC.
FIG. 9 shows the results of detection of differentiation of iMSC into adipocytes.
FIG. 10 shows the results of detection of differentiation of iMSC into osteoblasts.
FIG. 11 shows the results of detection of differentiation of iMSC into chondroblasts.
Fig. 12 shows the results of the pre-operative body weight and blood glucose measurements of mice: (a) body weight; (B) blood sugar.
Fig. 13 is a mouse wound treatment model: (a) plucking of the back hair of the mice; (B) marking of the excision site 1.5X1.5 cm on the back of the mouse; (C) Excision of the mouse back full cortex and site of cell injection; (D) measuring the initial resection area.
Fig. 14 is a real photograph of a wound of a mouse after surgery.
Fig. 15 is a graph of wound healing efficiency after mice surgery.
Fig. 16 shows the blood glucose test results after the mice operation.
FIG. 17 shows H & E staining results of wound tissue after 14 days post-surgery in mice, with boxes for magnification sites, 1-fold, 5-fold, and 10-fold in order from left to right.
Fig. 18 is the wound tissue healing results 14 days after mice surgery.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples, which do not represent limitations on the scope of the present invention. Some insubstantial modifications and adaptations of the invention based on the inventive concept by others remain within the scope of the invention.
Description of the terms
The term "pluripotent stem cells (pluripotent stem cell, PSC)" as used herein refers to a class of cells that possess both self-renewing and multipotent differentiation potential, including but not limited to: (1) Embryonic-derived pluripotent stem cells, (2) somatic cell reprogramming resulting pluripotent stem cells.
The term "induced pluripotent stem cells (ipscs)" as used herein refers to a class of stem cells that the somatic cells develop back into a state of multipotent differentiation potential via a reprogramming process. The multi-energy expression mode is as follows: has the ability to differentiate into three germ layers and multiple cell types.
The term "differentiation" as used herein refers to the process by which stem cells are transformed into a cell type of interest in a specific context.
The terms "iMSC" and "iMSCs" as used herein are used interchangeably herein to refer to the induced mesenchymal stem cells (induced-mesenchymal stem cells) of the present invention.
EXAMPLE 1 cultivation of iPSC
The iPSC of this example was purchased from the well traceability company under the product number RC01010.
(1) The day before resuscitating the cells, 1.5ml matrigel was placed in a 30mm dish, placed in an incubator and allowed to stand overnight at 37 ℃.
(2) Resuscitating: taking out the frozen iPSC from the liquid nitrogen tank, placing the iPSC in a water bath kettle at 37 ℃, and taking out when ice cubes in the frozen tube are melted to the size of mung beans. The cells in the cryopreservation tube were transferred to a centrifuge tube containing 5ml of TeSR-E8 medium, centrifuged at 500g for 5min at room temperature and the supernatant was aspirated off. 2ml of PBS was added, centrifuged at 500g at room temperature for 5 minutes, and the supernatant was aspirated off.
(3) Culturing: cell pellet was resuspended in 1mLTeSR-E8 medium and Mat was aspirated from a 30mm dishAfter the rigel liquid, the resuspended cells were uniformly inoculated into a petri dish and placed at 37℃in 5% CO 2 Is cultured in a cell culture incubator. The culture solution is replaced every 24 hours, the cell state is observed, and after the cell fusion degree reaches 80% -90%, the cell is subjected to passage.
(4) And (3) passage: the medium was discarded, cells were washed 2 times with pre-warmed PBS, the residual solution was aspirated, 2ml of PBS solution containing 5mM EDTA was added, and the solution was digested in an incubator for 3-5 minutes, and the digested solution was aspirated when the cell pellet was loose and allowed to leave the dish. An appropriate amount of E8 medium was added and the cells gently blown. Cells were pipetted into a 15ml centrifuge tube, centrifuged at 300g for 5 minutes at room temperature and the supernatant was pipetted off. Resuspension of cells with E8 medium, uniform inoculation of resuspended cells into six well plates pre-plated with Matrigel, and placement at 37deg.C, 5% CO 2 Is cultured in a cell culture incubator.
EXAMPLE 2 Induction and culture of iMSCs
1. Induction of iMSCs
The composition of the medium used in the induction process is shown in Table 1.
TABLE 1 Medium composition
Figure BDA0003975796200000061
Figure BDA0003975796200000071
(1) After the iPSC of example 1 had grown for 48 hours on the wall, the medium was aspirated, medium A was added, and the culture was continued for 24 hours at 37℃under 5% CO 2.
(2) Completely sucking and removing the culture medium A, adding the culture medium B, and culturing normally for 24 hours.
(3) Completely sucking and removing the culture medium B, adding the culture medium C, and culturing normally for 48 hours.
(4) Completely absorbing and removing the culture medium C, washing 2 times by using PBS, adding tryple to digest cells, re-inoculating the cells into a plate paved with 0.1% gelatin coating solution, adding the culture medium D, and culturing normally for 48 hours.
(5) Half of the volume of medium D was aspirated, half of the volume of medium E was added, and the culture was continued for 24 hours.
(6) Completely sucking and removing the culture medium in the step (5), adding fresh culture medium E, continuously inducing and differentiating to 90% density (about day 10), adding tryple digestive cells, inoculating to the culture medium F, continuously culturing for 14 days, and recording the obtained iMSCs as P0 generation.
As shown in fig. 1, on the first day after the addition of medium a, a floating phenomenon of a part of ipscs was observed under a microscope, and the floating ipscs were removed by a liquid exchange method. The third day a clear change in cell morphology was observed under the microscope, cells at the edges of the cell colonies exhibited a clear long fusiform shape, but cells located in the middle part of the cell pellet also contained morphological features of ipscs. Only cells in spindle-like morphology were observed under the microscope in the eighth day. Cells in the form of typical spindles were observed under a microscope on the twelfth day in such a manner that they grew in an adherent manner and the cells grew normally. Has grown full and can be used for subculture.
2. Cultivation of iMSC
(1) The medium used for the generation P0 iscs was discarded, the cells were rinsed with PBS, digested with TrypLE, after cell shrinkage was observed under a microscope, medium F was added in twice the volume of the digest, the digestion reaction in the dish was stopped, and the digested cells were collected into a centrifuge tube with a pipette.
(2) Centrifuging for 5min at 300rcf, discarding supernatant, re-suspending cells with PBS, centrifuging for 3 min at the same speed, discarding supernatant;
(3) The culture medium F is selected to resuspend the cells, and the proper proportion is selected for subculture according to the growth speed of the cells.
(4) Observing the growth condition of the cells, and periodically replacing the culture medium or passaging again to ensure that the cells have healthy growth conditions.
FIG. 2 shows the cell morphology of the iMSCs of the P0, P6 and P10 generations. The observation under microscope shows that the cell morphology of the iMSCs is stable and consistent, and the iMSCs are all typical spindles, and the growing mode is adherent growth and the cell growth state is normal.
EXAMPLE 3 identification of iMSC
1. iMSC surface molecular identification
The characteristic surface markers of the P6 generation iMSC in example 2 were examined using a flow cytometer (FIG. 3), and the results showed that the iMSC expressed CD73, CD90 and CD105 (. Gtoreq.95%), did not express CD14, CD34, CD45, CD79a and HLA-DR (. Gtoreq.5%), and met the MSC standard. Meanwhile, 97.3% of the iMSCs express CD73, CD90 and CD105 simultaneously, which suggests that the iMSCs induced by the invention have stronger dryness.
2. Security authentication
(1) Subcutaneous tumorigenicity assay
a. The P5 generation ifscs of example 2 were collected. Meanwhile, collecting UC-MSCs, wherein the separation method of the UC-MSCs refers to CN113403271A, and inoculating the separated UC-MSCs in a culture medium F for passaging, and collecting P5 generation UC-MSCs.
b. Raising mice: SPF grade healthy 6-week-old immunodeficiency NOD-SCID female mice purchased from Beijing velocide China are selected, the mice are bred in a stable environment with the temperature (20+/-2) DEG C and the humidity of 50-60%, 12h light and 12h dark environments are alternately provided every day, water is freely fed, and the mice are bred in animal houses for one week.
c. Subcutaneous injection: after one week, the backs of the mice were grasped to expose the underarm inoculation sites, and the group of iMSCs mice was inoculated with 100. Mu.L of iMSCs (about 2X 10 6 Individual/individual); mice of the UC-MSCs group were vaccinated with 100. Mu.L of UC-MSCs (about 2X 10) 6 And/or just).
The preparation method of the iMSCs cell PBS suspension comprises the following steps: the P5 generation iMSC is collected, washed by PBS, digested by TrypLE, centrifuged for 5 minutes at a rotation speed of 300rcf, the cell pellet is collected, resuspended by PBS, and screened to prepare single cell suspension, and the cell suspension is adjusted to a proper concentration. The preparation method of the UC-MSCs cell PBS suspension is the same as the above.
When in inoculation, the needle is inserted from the part of the mouse, which is slightly above the waist, so that the distance between the needle and the inoculation point is smaller than the length of the needle head, and the needle head passes towards the head part, so that the muscle layer cannot be pierced. Mice were kept on after inoculation, and the subcutaneous tumor-bearing status of both groups of mice was observed periodically.
After 2 months of feeding, as shown in FIG. 4, no tumor tissue was detected at the cell injection site of both NOD-SCID mice injected with UC-MSCs and iMSCs.
(2) in vivo return detection of iMSCs
a. Raising mice: NOD-SCID mice of 8 weeks old purchased from Beijing veloci-Hua laboratory animal company are selected, the mice are bred in a stable environment with the temperature (20+/-2) ℃ and the humidity of 50-60%, 12h illumination and 12h dark environments are alternately provided daily, water is freely fed for eating, and the mice are adaptively bred in animal houses for one week.
b. The cells of example 2P6 generation iMSCs were collected, counted by digestion, and the cell suspension was adjusted to a suitable concentration, and the mice of the experimental group were injected intravenously at 5X 10 cells according to the body weight of the mice 6 cells/kg. A549 cells (available from Nanjing, bai Biotechnology Co., ltd., product number CBP 30021L) were injected subcutaneously into the forelimb of control mice at a cell mass of 2X 10 6 And/or just.
c. Preparing luciferase stock solution with concentration of 30mg/mL with sterile water, gently turning over to dissolve luciferin, packaging, and storing at-20deg.C. Before use, the storage solution is diluted by PBS to a final concentration of 150 mug/mL, and the mice in the experimental group and the control group are respectively injected with luciferase with a volume of 200 mug in the abdominal cavity, and after injection, the mice are transferred to a gas hemp warehouse and then are mechanically detected to be distributed in animal bodies after 10min. And performing in-vivo imaging at 1h, 4h, 8h, 24h and 48h after injection.
The results showed that larger tumor masses appeared in mice injected with tumor cells A549-hluc (FIG. 5). In mice injected with iMSCs by tail vein, cells quickly home to the lung of the mice within 1 hour, the mice transfer to the intestines 4 hours after injection, and fluorescent signals of the iMSCs are basically not detected in the mice after 48 hours (figure 6), so that the safety of the iMSCs after implantation in the body is further proved.
(3) Detection of expression of pluripotent factors
The following operations were performed with iPSC cultured in example 1 and P6 generation imascs cultured in example 2, respectively:
1) Extraction of total RNA in tissue:
a. after the culture broth was aspirated, rinsed with PBS, 10ml of Trizol was added, and the cells were lysed by repeated pipetting with a pipette, transferred to a centrifuge tube, and centrifuged at 1000g for 1 min.
b. After centrifugation, transferring the Trizol lysate of the upper layer into a new centrifuge tube, and standing for 5min at room temperature;
c. chloroform was added to the centrifuge tube in a proportion of 0.2mL chloroform per 1mL of LTril, vortexed for 15-30s, and after thoroughly mixing, the mixture was allowed to stand at room temperature for 30min, and centrifuged at 4℃for 15min at 12000rpm.
d. The supernatant after centrifugation was aspirated, transferred to a new centrifuge tube, added with isopropanol twice the volume of the supernatant, allowed to stand at room temperature for 10min, centrifuged at 4℃for 10min at 12000rpm, and the supernatant was discarded.
e. To the centrifuge tube, 1mL of 75% ethanol was added to wash the pellet, centrifuged at 5min at 4℃at 7500rpm, and the supernatant was discarded.
f. Sucking up the liquid in the tube as much as possible, naturally drying the precipitate at room temperature, and dissolving the precipitate in Rnse-free ddH 2 O。
g. The concentration of RNA is measured, marked and frozen in a refrigerator at-80 ℃ for standby.
2) RNA reverse transcription cDNA:
the reverse transcription kit (abclon al, RK 20428) was used as follows:
the reaction system for reverse transcription is as follows:
a.2. Mu.L of sample RNA (500 ng/. Mu.L), 2. Mu.L of BuLge-Loop TM miRNA RT-primer(500nM)、Rnase free ddH 2 O was made up to 12. Mu.L. Mixing, centrifuging briefly, water-bathing at 70deg.C for 10min, and rapidly placing on ice for 2min.
b. mu.L of 5 XM-MLV buffer, 1. Mu.L of dNTP (2.5 nM), 0.5. Mu. L RNase inhibitor (40U/. Mu.L), 1. Mu.L of RTase M-MLV (200U/. Mu.L), 1.5. Mu.L of Nuclease free water were added, and the total amount was 20. Mu.L. The reverse transcription reaction conditions were as follows: 30min at 42 ℃ and 15min at 70 ℃, and rapidly placing on ice after the reaction is finished.
3) qRT-PCR detection:
BIO-RAD Real-time fluorescence quantitative PCR instrument is selected, real-time PCR primer is designed and synthesized by Huada biological company, and specific sequence is shown in table 3.
TABLE 3 PCR primer sequences
Figure BDA0003975796200000091
Figure BDA0003975796200000101
The PCR reaction system is shown in Table 4.
TABLE 4 PCR reaction System
Composition of the reaction System Volume of
cDNA 2μL
2×SYBRgreenqPCRmix 10μL
qPCR-primer-F 1μL
qPCR-primer-R 1μL
Nuclease 6μL
Total volume of 20μL
Reaction conditions: 95 ℃ for 10min; the temperature of 95 ℃ is 30s, the temperature of 60 ℃ is 30s as one cycle, and 40 cycles are taken.
The relative expression levels of OCT4, NANOG, SOX2 and KLF4 were calculated by delta-delta Ct method using GAPDH as an internal reference, and the difference between the expression levels of iPSC and iMSCs was compared.
As shown in FIG. 7, iPSC expressed OCT4, NANOG, SOX2, KLF4 normally, iMSCs did not detect OCT4, NANOG, SOX2, KLF4 expression, suggesting that the induction process was more complete and the purity of the resulting cell line was higher.
(4) Telomerase activity assay
Telomerase activity was detected by telomere repeat amplification (Telomerase repeat amplification protocol, TRAP) and the telomerase activity TRAP real-time quantitative detection kit was purchased from Shanghai halin biosystems, inc. Under the accession number HL20106.2. The P6 generation iMSC in example 2 was selected as the experimental group, the 293 cell line (purchased from the national experimental cell resource sharing platform, cat No. 1101HUM-PUMC 000010) was selected as the positive control group, and the lysate without the addition of the sample was selected as the negative control group. The reactivity is high or low according to the Ct value.
According to instruction, sample splitting and protein identification are sequentially carried out to adjust the concentration of tissue splitting liquid and the analysis of TRAP on-machine. The total reaction system is 25 mu L, the reaction solution B, the dyeing solution C and the supplementing solution D are sequentially added, then the sealing solution is added into the reaction system for placing the system for volatilization after short centrifugation, and then the PCR detection is carried out on the system by a machine, wherein the reaction conditions are as follows: 20min at 30 ℃; the amplification was carried out in 35 cycles at 95℃for 30s and 60℃for 90 s.
The results are shown in FIG. 8, and the results of the iMSC telomerase activity detection are negative, which shows that the iMSC telomerase activity detection has better safety (no tumorigenicity).
3. Induced differentiation capability assay
(1) iMSC adipogenic cell differentiation capability detection
The P6 generation iMSC of example 2 was selected and the lipid-forming induced differentiation kit was purchased from Cyagen (cat No. HUXMA-90031).
1) Pretreatment: sucking 1ml of 0.1% gelatin into six-hole plate, slowly shaking to uniformly cover the bottom surface of the isolation hole, and spreading 0.1% gelatin into six-hole platePlaced at 37℃with 5% CO 2 Is allowed to stand for 1 hour in the incubator. iMSC of generation P6 was placed at 37℃and 5% CO 2 Is digested with TrypLE when the cell fusion reaches 80-90%.
2) Inoculating: sucking out the gelatin in the hole, and mixing the digested P6 generation iMSC cells according to 2×10 4 cells/cm 2 Is inoculated into a six-well plate, 2mL of complete medium (medium F described in example 2) is added to each well, and the mixture is placed in an incubator at 37℃and 5% CO2 for culturing. The solution was changed every 1 day until the cell fusion reached 100%.
3) Induction: carefully aspirate the complete medium from the well and add 2mL of adipogenic differentiation medium a to a six well plate
After three days of induction, the solution A in the six-hole plate is sucked, 2mL of solution B of the adipogenic differentiation medium is added, after 2 days of maintenance, the solution B is sucked, and the solution A is replaced for continuous induction. Fluid A and fluid B were alternately changed and the induction and maintenance process was repeated three consecutive weeks.
4) Fixing: after completion of adipogenic differentiation, the medium in the six well plates was aspirated, and the well plates were gently washed 2 times with 1 XPBS. 2mL of 4% paraformaldehyde solution was added to each well and the mixture was fixed for 30min.
5) Dyeing: sucking the fixing solution, gently washing the pore plate with 1 XPBS solution for 2-3 times, washing the fixing solution thoroughly, adding 2mL of prepared oil red O dye solution working solution (working solution preparation method: oil red O stock solution: distilled water) into each well
=3:2, mixed well and filtered with neutral filter paper), stained for 30min at room temperature.
6) Cleaning: the oil red O staining solution is sucked and removed, the solution is gently washed for 2 to 3 times by using 1 XPBS solution, after the staining solution is fully washed, 2mL of 1 XPBS solution is added to each hole, and the culture plate is placed under a microscope to observe the fat staining effect.
The results are shown in fig. 9, and after lipid induction for 21 days, a clear red lipid drop was observed in the visual field using oil red O staining, indicating that the iMSC has the capacity to adipogenic differentiation.
(2) iMSC osteoblast differentiation capability detection
The osteogenic differentiation kit was purchased from Cyagen (HUXUC-90021) using the P6 generation iMSC of example 2.
1) Pretreatment: sucking 1mL of 0.1% gelatin into six-hole plate, shaking slowly to cover the bottom surface of the isolation hole uniformly, placing six-hole plate coated with 0.1% gelatin at 37deg.C and 5% CO 2 Is allowed to stand for 1 hour in the incubator. iMSC of generation P6 was placed at 37℃and 5% CO 2 Is digested with TrypLE when the cell fusion reaches 80-90%.
2) Inoculating: sucking off the gelatin in the wells, and subjecting the digested iMSCs of the P6 generation to a procedure of 2X 10 4 cells/cm 2 Is inoculated into a six-well plate, 2mL of complete medium (medium F described in example 2) is added to each well, and the mixture is placed in an incubator at 37℃and 5% CO2 for culturing. The solution was changed every 1 day until the cell fusion reached 70%.
3) Induction: the complete medium in the wells was carefully aspirated, 2mL of osteogenic differentiation medium was added to the six well plate, the liquid was changed every 2 days, and induction was continued for 3 weeks.
4) Fixing: after completion of osteogenic differentiation, the osteogenic differentiation complete medium in the six well plates was aspirated, and 1 XPBS was used
The well plate was gently washed 2 times. 2mL of 4% paraformaldehyde solution was added to each well and the mixture was fixed for 30min.
5) Dyeing: the fixative was aspirated, the well plate was gently washed 2-3 times with PBS solution, after which 2mL alizarin red working solution was added to each well and stained 5min at room temperature.
6) Cleaning: the alizarin red staining solution is sucked off, the solution is gently washed for 2 to 3 times by using a 1 XPBS solution, 2mL of the 1 XPBS solution is added to each hole after the staining solution is fully washed off, and the culture plate is placed under a microscope to observe the osteogenic staining effect.
The results are shown in FIG. 10, and as a result of the experiment after 24 days of osteoinduction, a large number of calcium nodules were observed and exhibited a red color.
(3) iMSC chondroblast differentiation capability detection
The P6 generation iMSC of example 2 was selected and the chondrogenic differentiation kit was purchased from Cyagen (HUXUC-90041).
1) Pretreatment: will be 4×10 5 The P6 generation iMSC cells were transferred to a 15mL centrifuge tube, centrifuged at 300 Xg for 4min at room temperature, and the supernatant was discarded. To the cell pellet was added 0.5mL of the chondrogenic differentiation premix, the cell pellet was resuspended, centrifuged at 150 Xg for 5min at room temperature, and the washing was repeated once.
2) Preparing a cartilage induced differentiation complete culture medium: 10 mu LTGF-beta 3 is added into 1mL of the cartilage-forming induced differentiation premix, and the mixture is gently blown and mixed. The complete culture medium for inducing and differentiating the cartilage is prepared at present and stored at 4 ℃ for no more than 12 hours.
3) Inoculating: the cell pellet obtained in step 1) was resuspended in 0.5mL of chondrogenic induced complete differentiation medium and centrifuged at 150 Xg for 5min at room temperature, and the centrifuge tube was unscrewed for gas exchange. Standing at 37deg.C with 5% CO 2 CO at saturation humidity 2 Culturing in an incubator.
4) Induction: when the aggregation phenomenon of cells is observed in the next day, the bottom of the centrifugal tube is flicked, and the cartilage balls are separated from the bottom of the centrifugal tube and are suspended in the liquid. Starting from inoculation, fresh chondrogenic differentiation complete medium is replaced to cells every 2 days, and induction is continued until cartilage balls with the diameter of 1.5-2mm are formed in the tube, so that sections can be prepared for staining.
5) Fixing: the cartilage balls thus induced were washed with 1 XPBS, immersed in a 4% paraformaldehyde solution, and fixed for 1 hour.
6) Dehydrating: the immobilized cartilage balls were dehydrated with gradient concentration alcohol, and the dehydration procedure is shown in Table 5.
TABLE 5 gradient dehydration procedure
Alcohol concentration Processing time
50％ 30min
70％ 30min
80％ 30min
95％ 30min
100％ 30min
7) And (3) transparency: gradient transparent treatment with xylene after dehydration:
a. mixing xylene and absolute alcohol at volume ratio of 1:1, and soaking cartilage ball for 2 hr.
b. The cartilage pellet was soaked in pure xylene for 1.5h.
c. The fresh xylene was replaced and the soaking was continued for 1h.
In the process of transparency, if white vaporific bubbles appear around the cartilage ball, the cartilage ball should be put back into alcohol again for dehydration and transparency again.
8) Wax dipping: soaking cartilage balls in a 40 ℃ solution prepared by uniformly mixing dimethylbenzene and paraffin in a volume ratio of 1:1 for 40min, and then transferring the cartilage balls into liquid paraffin, and soaking for 1h at a constant temperature of 55 ℃.
9) Embedding: and taking out the cartilage ball, placing the cartilage ball in a mould, pouring paraffin, standing and cooling. And after the paraffin is fully cooled and formed, taking out and correcting the paraffin block.
10 Slice): slicing by a slicer, adhering the slices of the cartilage balls on an adhesive glass slide, fishing out the slices, baking and storing for later use.
11 Dewaxing): the slice is soaked in pure xylene for 15min, and a new slice is soaked in xylene for 10min. The dimethylbenzene and the absolute alcohol are uniformly mixed according to the volume ratio of 1:1. Soaking the slices for 10min. Sequentially soaking the slices in 95%, 85%, 70% and 50% alcohol for 10min, and air drying.
12 Dyeing: the slice was stained with aliskirin blue for 1 hour at room temperature, and the surface was rinsed with tap water to observe the effect of aliskirin blue staining under a microscope.
As a result, the iMSC can form a structurally intact cartilage sphere as shown in FIG. 11.
The results show that the iMSC obtained by induction culture has good three-line differentiation capacity.
EXAMPLE 4 repair of skin lesions in diabetic mice by iMSC
1. Adaptive feeding of diabetic mice
Female diabetic mice were selected from 8 week old International model mice db/db from Hangzhou child source laboratory animals. Mice were fed in animal rooms, alternately providing 12h light and 12h dark environments daily, at 25 ℃, with free water intake for feeding. After one week of feeding, the mice were randomly divided into a Blank Control group (Blank), a negative Control group (Control), a UC-MSCs treated group and an iscs treated group, four groups of four each.
2. Preoperatively measuring blood glucose and body weight of mice in each group
The blood glucose level and the body weight of the mice were measured daily before the operation, wherein the blood glucose level of the mice was measured as follows:
(1) Tail tip blood collection: the method comprises the steps of taking alcohol cotton to gently wipe the tip of a mouse tail, rotating to pull out a needle cap of a blood taking needle, carefully pressing the needle cap towards the tail end of the mouse, and after the tail tip oozes out a round blood drop, enabling the top end of blood glucose test paper to be close to a blood sample.
(2) Blood glucose test: the contact strip is upwards, and the test paper is inserted into the inlet of the glucometer to wait for reading. And after the detection is finished, the test paper is taken down, and the reading value is recorded.
The basic features of the four groups of mice are shown in figure 12. After random grouping, the appearance morphological characteristics of the mice remained substantially consistent, with the appearance of increased water intake and the basic characteristics of diuresis. The body weight of each group of mice was floated within the interval of (43.945.+ -. 9.65) g/L, and the blood glucose level of each group of mice was floated within the interval of (28.68.+ -. 4.5) mmoL/L, and the whole was within the range of higher blood glucose level. The weights and the blood sugar values of the mice in each group after grouping have no statistical difference, meet the basic experiment requirements, and can be used for carrying out subsequent experiments.
3. Construction of Back skin loss model of diabetic mice
The skin injury repair is carried out by adopting an operation mode of the full cortex excision of the back of the mouse and adopting an in-situ fixed-point injection mode. The specific operation steps are as follows:
(1) Anesthesia: the mice were given general anesthesia by intraperitoneal injection, fasted for 12 hours before surgery, the weights of the mice were weighed, and then injection was performed at a dose of 0.2mL/10 g body weight. The anesthetic was aspirated with a 1mL syringe, the internal air bubbles were evacuated, the mouse was grasped, turned over so that it was exposed to the abdominal cavity, the lower left abdomen of the mouse was kept away from the liver and abdominal bladder as a puncture point, the angle was adjusted after the needle tip was penetrated horizontally into the skin, the anesthetic was pushed in with the needle tip at 45 ° to the abdomen, the needle tip was withdrawn, the mouse was laid flat for about 5 minutes, and the subsequent operation was performed.
(2) Skin preparation: the back hair of the mouse is plucked, a square with the back side length of 1.5cm is measured by a steel ruler as a excision site, then the position needing excision is marked by a marker pen, and the back skin is wiped by iodophor for disinfection.
(3) Excision: in an operating table, full cortex excision of the back skin of the mouse is carried out along the marked graph by utilizing an ophthalmic scissors, an open wound surface avoidance valve and a venous great vessel are constructed, the incision size is measured again by using a steel rule, and the initial excision area is recorded.
(4) Injection: the injection is sucked by a 1mL syringe, four-point injection is carried out along the opening, after the mice are awake, single-cage feeding is carried out, and the mice maintain obese body type in the feeding process, and are accompanied with the conditions of increased water intake and urine volume.
The four groups of mice were injected as follows:
blank control group: no injection treatment was performed;
negative control group: four-point injection of PBS;
UC-MSCs treatment group: four-point injection of UC-MSCs (about 4X 10) 5 Individual/individual);
hmscs treatment group: four-point injection of iMSC (about 4X 10) 5 And/or just).
Cells used for injection: the iMSC is selected from the generation P6 iMSC in example 2; isolation of UC-MSC reference CN113403271A, and isolated UC-MSCs were inoculated in medium F for subculture to P6 generation. After the collected cells are digested and centrifuged, the cells are resuspended by PBS solution and are screened to prepare single cell suspension, and the cell suspension is adjusted to proper concentration. The injection amount of each group of cells is consistent and is 4 multiplied by 10 5 The total volume of injections was 50. Mu.L/dose.
The key procedure in the operation is shown in FIG. 13, where the arrows indicate the injection sites of cells (or equal volumes of PBS).
(5) Measurement: the blood glucose value and wound injury area of the mice were measured every other day.
(6) Two weeks later, mice were sacrificed by cervical scission.
4. Evaluation index
(1) Wound healing conditions:
wound defect areas of four groups of mice were measured on postoperative days 3, 7, 9, 11 and 13 (fig. 14), and wound healing efficiencies were calculated by counting the wound defect areas measured within 14 days, and a wound healing curve was drawn (fig. 15).
The results showed that the four groups of mice had no significant difference in wound defect area on the third day post-surgery. On the 7 th day after operation, the wound defect area of mice in the UC-MSCs treatment group and the iMSCs treatment group is smaller than that of the control group, and the wound defect area of the mice in the UC-MSCs treatment group and the iMSCs treatment group shows a trend of obviously shrinking along with the extension of time, and the defect area is smaller than that of the mice in the two control groups, so that the iMSCs are indicated to have tissue repair effects similar to those of the UC-MSCs, and the healing speed of ulcers of the diabetic mice can be accelerated.
(2) Blood glucose condition:
blood glucose values were measured for four groups of mice on postoperative days 1, 3, 7, 9, 11 and 13, as shown in fig. 16. The results indicated that on day 3 post-surgery, blood glucose levels were down-regulated in mice in the UC-MSCs and iMSCs treated groups. On postoperative day 5, blood glucose levels were up-regulated in mice in the UC-MSCs and iMSCs treatment groups. On the 7 th postoperative day, the blood glucose levels of mice in the UC-MSCs treatment group and the iMSCs treatment group are restored to the preoperative level, and then the blood glucose levels of the mice in the two experimental groups are gradually leveled with those of the mice in the two control groups for seven days, so that the blood glucose levels of the diabetic mice can be reduced in a short time by injecting the iMSCs.
(3) Pathology evaluation:
after two weeks post-surgery, four groups of mice were sampled for back injury sites, fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, hematoxylin and eosin stained (H & E stained), and pathology study was performed.
As shown in FIG. 17, compared with the Blank control group (Blank), the sections of the UC-MSCs treatment group and the iMSCs treatment group have similar repair results, the wound healing of the damaged part is more complete, the formation of new granulation tissues and new skin appendages at the damaged part can be observed, and the connection between the wound and the skin at the undamaged part is more complete.
After two weeks post-surgery, the area of the back neotissue of the mice was measured, and the wound healing area was calculated, and the results are shown in fig. 18, which indicate that the UC-MSCs treated group and the ifcs treated group can increase the ulcer tissue healing rate.
The results show that the iMSCs obtained by the induction culture of the invention have tissue repair capability similar to UC-MSCs.

Claims (10)

1. A culture medium combination, characterized in that the culture medium combination comprises a culture medium a, a culture medium B, a culture medium C, a culture medium D and a culture medium E;
the culture medium A is a culture medium in which BMP4 and activin A are added into a complete culture medium;
the culture medium B is a culture medium in which BMP4 is added into a complete culture medium;
the culture medium C is a culture medium in which ALK inhibitor is added into a complete culture medium;
the culture medium D is a culture medium in which bFGF is added to a complete culture medium;
the culture medium E is a culture medium obtained by adding FBS, VEGF, EGF, IGF-1, TGF-beta and bFGF into a basic culture medium;
the ALK inhibitors include SB431542.
2. The combination of media of claim 1, wherein the complete media comprises E6 media and E8 media;
preferably, the complete medium is E6 medium;
preferably, the basal medium comprises DMEM/F12, DMEM, a-MEM and knockoutDMEM/F12;
preferably, the basal medium is DMEM/F12.
3. The combination of claim 1, wherein BMP4 in medium a is 0.5-50 μg/L, activin a is 0.5-30 μg/L, BMP4 in medium B is 0.5-30 μg/L, SB431542 in medium C is 0.5-10uM, bFGF is 0.5-10 μg/L, fbg is 2-15%, VEGF is 0.5-10 μg/L, EGF is 0.5-10 μg/L, IGF-1 is 0.5-10 μg/L, TGF-B is 0.5-10 μg/L, bFGF is 0.5-10 μg/L;
preferably, the BMP4 concentration in the culture medium A is 5-25 mug/L, the activin A concentration is 5-25 mug/L, the BMP4 concentration in the culture medium B is 5-25 mug/L, the SB431542 concentration in the culture medium C is 1-10 mug/L, the bFGF concentration in the culture medium D is 1-10 mug/L, the FBS concentration in the culture medium E is 2-10%, the VEGF concentration is 1-10 mug/L, EGF concentration is 1-10 mug/L, IGF-1 concentration is 1-10 mug/L, TGF-B concentration is 1-10 mug/L, bFGF concentration is 1-10 mug/L;
preferably, the BMP4 concentration in the culture medium A is 5-20 mug/L, the activin A concentration is 5-20 mug/L, the BMP4 concentration in the culture medium B is 5-20 mug/L, the SB431542 concentration in the culture medium C is 2-8 mug/L, the bFGF concentration in the culture medium D is 2-8 mug/L, the FBS concentration in the culture medium E is 2-8%, the VEGF concentration is 1-8 mug/L, EGF concentration is 1-8 mug/L, IGF-1 concentration is 1-8 mug/L, TGF-B concentration is 1-8 mug/L, bFGF concentration is 1-8 mug/L;
preferably, the BMP4 concentration in the culture medium A is 5-15 mug/L, the activin A concentration is 5-15 mug/L, the BMP4 concentration in the culture medium B is 5-15 mug/L, the SB431542 concentration in the culture medium C is 4-6 mug/L, the bFGF concentration in the culture medium D is 4-6 mug/L, the FBS concentration in the culture medium E is 4-6%, the VEGF concentration is 1-5 mug/L, EGF concentration is 1-5 mug/L, IGF-1 concentration is 1-5 mug/L, TGF-B concentration is 1-5 mug/L, bFGF concentration is 1-5 mug/L;
preferably, the BMP4 concentration in the culture medium A is 10 mug/L and the activin A concentration is 10 mug/L;
preferably, the BMP4 concentration in said medium B is 10. Mu.g/L;
preferably, the concentration of SB431542 in said medium C is 5uM;
preferably, the bFGF concentration in the medium D is 5. Mu.g/L;
preferably, the medium E has a FBS concentration of 5%, a VEGF concentration of 2. Mu.g/L, EGF, a 2. Mu.g/L, IGF-1, a 2. Mu.g/L, TGF-b concentration of 2. Mu.g/L, bFGF and a 2. Mu.g/L concentration.
4. The combination of media of claim 1, further comprising media F, wherein media F is media supplemented with FBS, VEGF, EGF, IGF-1, TGF-b, bFGF, p38MAPK inhibitor to basal media;
preferably, the p38MAPK inhibitor is SB202190;
preferably, medium F has a FBS concentration of 5%, VEGF concentration of 2. Mu.g/L, EGF, 2. Mu.g/L, IGF-1, 2. Mu.g/L, TGF-b, 2. Mu.g/L, bFGF, 2. Mu.g/L, SB202190 and 5. Mu.M;
preferably, the basal medium comprises DMEM/F12, DMEM, a-MEM, knockoutDMEM/F12;
preferably, the basal medium is DMEM/F12.
5. A method for preparing mesenchymal stem cells from multipotent stem cell differentiation, comprising the steps of:
(1) Culturing pluripotent stem cells to be induced by using a culture medium;
(2) Sucking and removing the culture medium in the step (1), adding the culture medium A, and continuing to culture;
(3) Completely sucking the culture medium A, adding the culture medium B, and continuing to culture;
(4) Completely sucking and removing the culture medium B, adding the culture medium C, and continuing to culture;
(5) Completely sucking and removing the culture medium C, adding the culture medium D, and continuing to culture;
(6) Sucking half of the volume of the culture medium D, adding half of the volume of the culture medium E, and continuing to culture;
(7) Completely sucking the culture medium in the step (6), adding a fresh culture medium E, and continuing culturing;
(8) And (5) digesting and passaging to a culture medium F when the cells grow fully, and continuing to culture to obtain the mesenchymal stem cells.
6. The method of claim 5, wherein the pluripotent stem cells comprise embryonic stem cells and induced pluripotent stem cells;
preferably, the pluripotent stem cells are induced pluripotent stem cells;
preferably, in the step (1), the culture medium of the pluripotent stem cells is selected from one of mTESR1 culture medium, teSR-E8 culture medium and Stemflex culture medium;
preferably, in the step (1), the culture medium of the pluripotent stem cells is TeSR-E8 culture medium.
7. The method of claim 5, further comprising passaging the mesenchymal stem cells obtained in step (6);
preferably, the passaging comprises inoculating the cells into medium F for culturing.
8. Mesenchymal stem cells obtained by the method of any one of claims 5-7.
9. The mesenchymal stem cell of claim 8, wherein the mesenchymal stem cell highly expresses CD73, CD90, CD105, while lowing expression of CD14, CD34, CD45, CD79a, and HLA-DR.
10. Use of the mesenchymal stem cells of any one of claims 8-9 in the preparation of a medicament for treating a disease;
preferably, the disease comprises wound repair;
preferably, the wound is a skin lesion;
preferably, the skin injury is a wound of a diabetic patient.
CN202211536629.9A 2022-12-01 2022-12-01 Culture medium combination, preparation method and application of mesenchymal stem cells Pending CN116426464A (en)

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