CN113652393A - Method for improving pluripotency of bovine embryonic stem cells - Google Patents
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Abstract
The invention provides a method for improving the pluripotency of bovine embryonic stem cells, which comprises the step of adding an antitumor drug naringenin with the final concentration of 10-40 mug/mL into a bovine embryonic stem cell in-vitro culture system for maintenance culture. The results of the pluripotency detection show that the naringenin can obviously enhance the expression of pluripotent factors of the bovine embryonic stem cells, and simultaneously, the naringenin can promote various original apparent forms of the bovine embryonic stem cell line to tend to be uniform. The method for in vitro culture of bovine embryonic stem cell pluripotency by using the antitumor drug naringenin provides bovine embryonic stem cells which can be used for gene transfection and in vitro long-term culture screening for research and production of transgenic cloned cows, and can be used for further research related to bovine embryonic stem cells.
Description
Technical Field
The invention relates to the field of cell biology and molecular biology, in particular to a method for improving the pluripotency of bovine embryonic stem cells.
Background
Embryonic Stem Cells (ESCs) are pluripotent Cells that are derived from the Inner Cell Mass (ICM) of the embryo before implantation, remain in an undifferentiated state and are capable of self-renewal, have the potential to differentiate into all Cells of the body, and can be used for research on early Embryonic development.
The existing method for establishing and maintaining the bovine embryonic stem cells has certain problems, such as unsatisfactory pluripotency, inhomogeneous phenotype, poor applicability and the like. Improve the pluripotency of the bovine embryonic stem cells, so that the bovine embryonic stem cells have wider applicable capability and have important biological significance.
Naringenin (Naringenin) with chemical name of 4,5, 7-trihydroxyflavone and molecular formula of C15H12O5Is a flavonoid compound widely existing in citrus fruits, tomatoes, cherries, grapefruits and other plants. Naringenin has been reported to have many pharmacological properties including anti-tumor, anti-cancer, anti-inflammatory, cardioprotective, antilipidemic, antiobesity, anti-diabetic, and anti-fibrotic, etc. And has wide biological activity, such as free radical scavenging activity, antioxidation, antiproliferation, etc. At present, no research report about the application of naringenin to the in vitro culture of bovine embryonic stem cells is found.
Disclosure of Invention
The invention aims to provide a method for improving the pluripotency of bovine embryonic stem cells.
In order to realize the purpose of the invention, in a first aspect, the invention provides an application of an antitumor drug naringenin in-vitro culture of bovine embryonic stem cells, wherein the structure of the compound naringenin is shown as a formula 1):
in a second aspect, the present invention provides a method for increasing the pluripotency of bovine embryonic stem cells, comprising: the in vitro culture and passage of bovine embryonic stem cells are carried out by adding naringenin (preferably 10. mu.g/mL or 40. mu.g/mL) to the in vitro culture solution of bovine embryonic stem cells at a final concentration of 10 to 40. mu.g/mL.
The bovine embryonic stem cells have concentration dependence on naringenin, the optimal long-term culture effect can be achieved when the naringenin concentration is 10 mu g/mL, and the pluripotent marker genes of the bovine embryonic stem cells are obviously up-regulated. When the concentration of naringenin is 40 mug/mL, the best short-term culture effect can be achieved, and the upregulation degree of the pluripotent marker gene of the bovine embryonic stem cell is higher than that of 10 mug/mL. When the concentration of naringenin is 60 mug/mL, the naringenin has stronger toxicity to bovine embryonic stem cells, the proliferation of the bovine embryonic stem cells is obviously inhibited, the up-regulation degree of the pluripotency marker gene of the bovine embryonic stem cells is lower than that of 40 mug/mL, but the up-regulation degree is obviously higher than that of a control group (bovine embryonic stem cells which are not cultured by naringenin). When the concentration of naringenin is 80 mug/mL, the naringenin has obvious toxicity to bovine embryonic stem cells.
The method for improving the pluripotency of the bovine embryonic stem cells comprises the steps that an in vitro culture solution contains 10-40 mu g/mL naringenin, 20ng/mL rhFGF2 (recombinant human fibroblast growth factor-2), 13.28mg/mL low fat acid BSA (low fatty acid bovine serum albumin), and 2.5 mu m of TeSR-E6 (TeSR-E6) IWR-1TM-E6) medium.
IWR-1 is Wnt pathway inhibitor with molecular formula of C25H19N3O3The structural formula is as follows:
in the method, the culture conditions for culturing the bovine embryonic stem cells in vitro are as follows: 37 ℃ and 5% CO2The culture medium was changed once a day. During the culture process, the cells are co-cultured with feeder layer cells obtained by mitomycin treatment of C57 mouse fetal fibroblasts.
In the present invention, the cell passaging method comprises: preparing a well plate paved with feeder cells (freshly prepared) in advance; bovine embryonic stem cells were digested with TripLE Select for 3min, and the digestion was stopped with DMEM/F12 basal medium at a passage ratio of 1: 4.
The preparation method of the feeder layer cells comprises the following steps: thawing primary C57 mouse fetal fibroblasts at 37 ℃ in 5% CO2Carrying out amplification culture under the condition until the cell density reaches 80-90 percent, treating the cells with a mouse fetal fibroblast culture solution containing 12 mu g/mL mitomycin for 2.5h, and freezing and storing the cells.
Wherein, the culture solution of mouse fetal fibroblast is: DMEM medium containing 10% FBS (fetal bovine serum) and 1% Penicilin-Streptomyces.
The method for using feeder layer cells comprises the following steps: after the cells are thawed and treated by the feeder layer one day before use and adhere to the wall overnight to restore the activity, the cells are co-cultured with the bovine embryonic stem cells.
The method for improving the pluripotency of the bovine embryonic stem cells further comprises the step of performing cryopreservation on the bovine embryonic stem cells which are cultured in vitro and passaged.
The cryopreservation method comprises the following steps: programmed cryopreservation was performed using the mFresR stem cell cryopreservation solution.
In the present invention, bovine ESCs are bovine embryonic stem cells obtained from bovine in vitro fertilized embryos.
The bovine embryonic stem cells obtained by the method for improving the pluripotency of the bovine embryonic stem cells can be stably passaged in vitro for a long time, and the cell clone morphology is uniform. The pluripotent property of the bovine embryonic stem cells is actually improved by the detection of various stem cell pluripotent means.
By the technical scheme, the invention at least has the following advantages and beneficial effects:
according to the invention, after the bovine embryonic stem cells are obtained by in vitro culture, the pluripotency of the bovine embryonic stem cells can be increased by adding the antitumor drug naringenin into a culture system for in vitro culture of the bovine embryonic stem cells, and the specific effective use concentration is 10-40 mug/mL. The bovine embryonic stem cells obtained by adding naringenin for in vitro culture can be subjected to conventional operations such as embryonic stem cell passage, cryopreservation, thawing and the like. The culture solution is required to be replaced once a day, and the passage ratio is 1: 4. And carrying out cell pluripotency identification on the bovine embryonic stem cells obtained by in vitro culture by adding naringenin in the passage process. Through multiple pluripotency tests, the conclusion is finally drawn that naringenin can obviously increase the expression of pluripotent factors of bovine embryonic stem cells, and meanwhile naringenin can promote various original apparent morphologies of bovine embryonic stem cell lines to be uniform. The method for in vitro culture of bovine embryonic stem cells by using the antitumor drug naringenin, disclosed by the invention, provides bovine embryonic stem cells which can be used for gene transfection and in vitro long-term culture screening for research and production of transgenic cloned cows, and can be used for further research related to the bovine embryonic stem cells.
The invention utilizes naringenin to improve the pluripotency of the bovine embryonic stem cells. The concentration dependence of the bovine embryonic stem cells on naringenin is specifically shown in the specification that the optimal long-term culture effect can be achieved when the naringenin concentration is 10 mu g/mL, the optimal short-term culture effect can be achieved when the naringenin concentration is 40 mu g/mL, the upregulation degree of the pluripotent marker genes of the bovine embryonic stem cells is higher than that of 10 mu g/mL, the naringenin concentration is 60 mu g/mL, the toxicity on the bovine embryonic stem cells is stronger, the proliferation of the bovine embryonic stem cells is obviously inhibited, the upregulation degree of the pluripotent marker genes of the bovine embryonic stem cells is lower than that of 40 mu g/mL, but the upregulation degree is obviously higher than that of a control group (bovine embryonic stem cells cultured without naringenin), and the toxicity on the bovine embryonic stem cells is obvious when the naringenin concentration is 80 mu g/mL, which indicates that the naringenin has obvious influence on the proliferation and the pluripotency of the bovine embryonic stem cells.
After the naringenin is treated, the expressions of the bovine embryonic stem cell pluripotency marker genes OCT4, SOX2 and NANOG are obviously up-regulated, the expressions of the initial state pluripotency marker genes (such as KLF4, DNMT3L, REX-1, TBX3 and the like) of the bovine embryonic stem cells are all obviously up-regulated, and the expressions of the initial state pluripotency marker genes (such as DNMT1, DNMT3A and DNMT3B) of the bovine embryonic stem cells are up-regulated in a small range, which shows that the naringenin can enable the pluripotency of the bovine embryonic stem cells to be greatly close to the initial state embryonic stem cells. The immunofluorescence staining result of the multi-functional marker genes such as SOX2, NANOG, SSEA4 and the like is positive, and the embryo-like bodies can be spontaneously formed in the basic fibroblast culture solution and can continue to be spontaneously differentiated.
Drawings
FIG. 1 shows the results of observation under a phase contrast microscope of bovine embryonic stem cells obtained after culturing with naringenin added in example 4 of the present invention (100X).
FIG. 2 shows the results of alkaline phosphatase staining of bovine embryonic stem cells obtained after incubation with naringenin in example 4 of the present invention (100X).
FIG. 3 shows the qPCR detection result of the relative expression change of the pluripotent gene of bovine embryonic stem cells obtained by adding naringenin and culturing in example 4 of the present invention.
FIG. 4 shows the qPCR detection result of the relative expression change of the initial pluripotent gene of bovine embryonic stem cells obtained after the cells are cultured by adding naringenin in example 4 of the present invention.
FIG. 5 shows the qPCR detection result of the relative expression change of the pluripotent gene in the initial state of the bovine embryonic stem cell obtained after the culture of the bovine embryonic stem cell added with naringenin in example 4 of the present invention.
FIG. 6 shows the results of immunofluorescent staining (100X) of bovine embryonic stem cells SOX2 obtained after culturing with naringenin added in example 4 of the present invention.
FIG. 7 shows the results of NANOG immunofluorescent staining of bovine embryonic stem cells obtained after incubation with naringenin in example 4 of the present invention (100X).
FIG. 8 shows the results of immunofluorescent staining (100X) of bovine embryonic stem cells SSEA4 obtained after culturing with naringenin in example 4 of the present invention.
FIG. 9 shows the result of immunofluorescent staining (100X) of bovine embryonic stem cells CDX2 obtained after culturing with naringenin added in example 4 of the present invention.
FIG. 10 shows the results of the spontaneous differentiation of bovine embryonic stem cells into embryoid bodies (100X) after the culture of example 4 of the present invention in which naringenin was added.
Detailed Description
The invention provides a method for improving the pluripotency of bovine embryonic stem cells, which is characterized in that under the condition of in vitro culture, 10-40 mu g/mL naringenin is added into a bovine embryonic stem cell culture system, so that the pluripotency of the bovine embryonic stem cells can be increased.
A culture method for improving the pluripotency of bovine embryonic stem cells comprises the following culture systems (10 mL):
in the method for improving the pluripotency of the bovine embryonic stem cells, the culture conditions are as follows: 37 ℃ and 5% CO2。
In the method for improving the pluripotency of the bovine embryonic stem cells, the in vitro culture of the bovine embryonic stem cells needs to be co-cultured with feeder layer cells obtained by treating in vitro cultured C57 mouse fetal fibroblasts.
The method for treating feeder layer cells obtained by in vitro culturing C57 mouse fetal fibroblasts is as follows: the mixture was treated with mitomycin 12. mu.g/mL for 2.5h and frozen. The using method of the feeder layer cells comprises the following steps: after the treated feeder layer cells are thawed overnight and adhered to the wall to restore the activity one day before use, the feeder layer cells can be co-cultured with the bovine embryonic stem cells.
In the method for improving the pluripotency of the bovine embryonic stem cells, the passage condition is TripLE Select digestion for 3min, and DMEM/F12 basic culture solution is used for stopping digestion, wherein the passage ratio is 1: 4.
In the method for improving the pluripotency of the bovine embryonic stem cells, the cryopreservation method is programmed cryopreservation by using mFreSR stem cell cryopreservation solution.
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.
The naringenin used in the following examples was purchased from Bailingwei technologies, Inc. of Beijing.
IWR-1 was purchased from Sigma Aldrich; y-27632 was purchased from STEMCELL; TeSRTM-E6 was purchased from stem cell; low fat acid BSA was purchased from MP Biomedicals; TripLE Select, Penicilin-Streptomyces from Gibico; mfsr from stemcel; IMDM, DMEM from Gibico; PBS, DMEM \ F12 was purchased from BI.
EXAMPLE 1C 57 preparation of mouse fetal fibroblast feeder layer
Thawing primary C57 mouse fetal fibroblasts at 37 ℃ in 5% CO2Amplification culture was performed to P3 under conditions, and after growth to a suitable density (80% -90%), treatment was performed for 2.5h with C57 mouse fetal fibroblast broth (DMEM + 10% FBS + 1% Penicillin-Streptomyces) containing 12. mu.g/mL mitomycin. After treatment, the mixture is frozen and stored in liquid nitrogen for later use. The application method comprises thawing mouse fetal fibroblast culture medium at 37 deg.C with 5% CO2Culturing overnight, washing twice with PBS, and replacing bovine embryonic stem cell culture solution for later use.
Example 2 acquisition and in vitro culture of bovine embryonic Stem cells
Removing zona pellucida from embryo of cattle blastocyst obtained by in vitro fertilization, inoculating to prepared mouse fetus fibroblast feeder layer, and culturing at 37 deg.C with 5% CO2And (5) culturing.
The culture system for in vitro culture of bovine embryonic stem cells is (10 mL):
the growth of the cells is observed after about 10 days of culture, and the bovine embryonic stem cells can be obtained after 4 generations of embryos grown from the cells (ICM cells). The passage condition is that TrypLE Select is digested for 3min, DMEM/F12 basic culture solution is used for stopping digestion, Rho kinase inhibitor Y-27632 is added into the culture system, and the culture system is changed into a culture system for in vitro culture of bovine embryonic stem cells after 24 h.
Culture system over 24h after passage (10 mL):
wherein the compound Y-27632 is a Rho kinase inhibitor with the molecular formula of C14H21N3O.2 HCl, the structure is shown as formula 2):
in vitro culture of bovine embryonic stem cells requires co-culture with feeder cells obtained from the treatment of fetal fibroblasts from in vitro cultured C57 mice.
During freezing, TripLE Select is used for digesting for 3min, centrifugation is carried out at 1500rpm for 5min, then supernatant is discarded, 500 mu L of mFreSR stem cell freezing solution is added, the mixture is transferred into a freezing tube and placed into a program freezing box, and the mixture is transferred into liquid nitrogen for preservation after being placed for 24h at minus 80 ℃.
When thawing, the freezing tube filled with bovine embryonic stem cells is quickly shaken in a water bath at 37 ℃ until all the freezing liquid is thawedAdding 1ml of DMEM/F12 basic culture solution into a freezing tube at room temperature, slightly blowing 3 times, sucking out the liquid into a centrifuge tube containing 5ml of DMEM/F12 basic culture solution, centrifuging at 1500rpm for 5min, sucking supernatant, resuspending cell precipitate with a subculture system of bovine embryonic stem cells, uniformly spreading the cell precipitate in a prepared 12-well plate (containing mouse fetal fibroblast feeder layer) at 37 ℃ and 5% CO2And (5) culturing. And after 24h, replacing the culture solution with a culture system of bovine embryonic stem cells.
Example 3A method for improving the pluripotency of bovine embryonic stem cells
Adding the antitumor drug naringenin into a bovine embryonic stem cell culture system, wherein the specific effective use concentration is 10-40 mu g/mL, the temperature is 37 ℃, and the CO content is 5 percent2The cell state is transformed and tends to be stable after 5 passages of culture.
The method for culturing the bovine embryonic stem cells in vitro by using naringenin uses a culture system (10 mL):
the passage condition is TrypLE Select digestion for 3min, and DMEM \ F12 basic culture solution is used for stopping digestion, and the passage ratio is 1: 4.
The bovine embryonic stem cells need to be co-cultured with the feeder layer cells, the feeder layer cells need to be unfrozen and attached to the wall one day before use, and can be co-cultured with the bovine embryonic stem cells after overnight vitality recovery. The freezing condition is as follows: digesting with TrypLE Select for 3min, centrifuging at 1500rpm for 5min, discarding the supernatant, adding 500 μ L of mFreSR stem cell frozen stock solution, transferring into a freezing tube, placing into a program freezing box, standing at-80 deg.C for 24h, and transferring into liquid nitrogen for preservation.
When thawing, rapidly shaking the freezing tube containing bovine embryonic stem cells in water bath at 37 ℃, rapidly adding 1ml of DMEM/F12 basic culture solution at room temperature into the freezing tube after the freezing solution is completely thawed, blowing 3 times slightly, sucking out the liquid into a centrifugal tube containing 5ml of DMEM/F12 basic culture solution, centrifuging at 1500rpm for 5min, completely sucking supernatant, resuspending cell sediment by using a culture system for in vitro culturing bovine embryonic stem cells containing antitumor drugs, and proportionally suspending the cell sediment by using the culture system for in vitro culturing bovine embryonic stem cells containing the antitumor drugsUniformly spread in a prepared 12-well plate (containing mouse fetal fibroblast feeder layer) at 37 deg.C with 5% CO2And (5) culturing.
The results of microscopic observation of bovine embryonic stem cells obtained after incubation with naringenin are shown in FIG. 1.
Example 4 identification of bovine embryonic stem cells obtained by transformation of bovine embryonic stem cells
1. Alkaline phosphatase staining
The staining procedure was followed using BCIP/NBT alkaline phosphatase color development kit (Beyotime, C3206) and bovine embryonic stem cells stained purplish red, indicating that alkaline phosphatase is expressed and that bovine embryonic stem cells are pluripotent (FIG. 2).
2. qPCR identification of stem cell pluripotency marker genes
The results of qPCR identification after reverse transcription of RNA of bovine embryonic stem cells obtained after treatment of bovine embryonic stem cells and naringenin with different concentrations (10. mu.g/mL, 25. mu.g/mL, 40. mu.g/mL and 60. mu.g/mL) are respectively extracted and are shown (FIG. 3-FIG. 5), after the bovine embryonic stem cells obtained after treatment of the antitumor drug naringenin, the expressions of the pluripotency marker genes OCT4, SOX2 and NANOG are all up-regulated, the expressions of the initial pluripotency marker genes (such as KLF4, DNMT3L, REX1 and TBX 3) of the bovine embryonic stem cells are all significantly up-regulated, and the expressions of the initial pluripotency marker genes (such as DNMT1, DNMT3A and DNMT3B) of the bovine embryonic stem cells are slightly up-regulated, which indicates that the naringenin can enable the bovine embryonic stem cells to approach the initial embryonic stem cells. The pluripotency of bovine embryonic stem cells is concentration dependent on naringenin. Wherein the effect is best when 40 mu g/mL of naringenin is used, but the effect is best when 10 mu g/mL of naringenin is used in the long-term culture process. The primer sequences used were as follows:
OCT4 upstream primer: 5'-GGTTCTCTTTGGAAAGGTGTTC-3'
A downstream primer: 5'-ACACTCGGACCACGTCTTTC-3'
SOX2 upstream primer: 5'-CATCCACAGCAAATGACAGC-3'
A downstream primer: 5'-TTTCTGCAAAGCTCCTACCG-3'
NANOG upstream primer: 5'-TTCCCTCCTCCATGGATCTG-3'
A downstream primer: 5'-ATTTGCTGGAGACTGAGGTA-3'
KLF4 upstream primer: 5'-TCCCACCGCTCCATTAC-3'
A downstream primer: 5'-ATGAGAACTCTTCGTGTAGG-3'
REX1 upstream primer: 5'-GGAAGAGGACCCACTCCTTC-3'
A downstream primer: 5'-ACTTGGCCTCCTAGTGCATC-3'
DNMT3L upstream primer: 5'-ATGAGCAACTGGGTCTGCTT-3'
A downstream primer: 5'-GGGCTCTCTCTTCCACACAG-3'
TBX3 upstream primer: 5'-CGGATTTACTTTGGCCTTCCC-3'
A downstream primer: 5'-CTGGTATGCAGTCACAGCGA-3'
Forward primer of TFCP2L 1: 5'-GTGCAGATCGACACCTTCAA-3'
A downstream primer: 5'-GGGAGCACTCTGAGAGGATG-3'
STELLA upstream primer: 5'-TGCAAGTTGCCACTCAACTC-3'
A downstream primer: 5'-TTCCTTTGGCATAGCGAAGT-3'
DNMT1 upstream primer: 5'-AGTGGGGGACTGTGTTTCTG-3'
A downstream primer: 5'-TGTACGAGAGCTGCATGTCC-3'
DNMT3A upstream primer: 5'-CTGGTGCTGAAGGACTTGGGC-3'
A downstream primer: 5'-CAGAAGAAGGGGCGGTCATC-3'
DNMA3B upstream primer: 5'-CCGCAGATCAAGCTCAC-3'
A downstream primer: 5'-GTTATTTCGGGTTCGGAC-3'
3. Immunofluorescence identification of stem cell pluripotent factors
And identifying the expression condition of the obtained bovine embryonic stem cell pluripotent factors. The results showed (FIGS. 6-9) that the obtained bovine embryonic stem cells expressed SOX2(Cell Signaling, L1D6A2), NANOG (Peprotech,500-P236), CDX2(sigma, AV31476) and SSEA-4(sigma, MAB 4304).
4. Differentiation of stem cells into embryoid bodies
The obtained bovine embryonic stem cells were cultured in IMDM medium containing 10% FBS and 1% Penicillin-Streptomyces for 7 days to obtain embryoid bodies of the bovine embryonic stem cells (FIG. 10).
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (8)
1. The application of the compound naringenin in-vitro culture of bovine embryonic stem cells, wherein the structure of the compound naringenin is shown in a formula 1):
2. a method of increasing the pluripotency of a bovine embryonic stem cell comprising: adding naringenin with the final concentration of 10-40 mug/mL into the in-vitro culture solution of the bovine embryonic stem cells for in-vitro culture and passage of the bovine embryonic stem cells; wherein the structure of naringenin is shown as formula 1).
3. The method of claim 2, wherein the in vitro culture fluid is TeSR-E6 medium containing 10-40 μ g/mL naringenin, 20ng/mL rhFGF2, 13.28mg/mL low fat acid BSA, and 2.5 μm IWR-1.
4. The method of claim 2, wherein the bovine embryonic stem cells are cultured in vitro using culture conditions selected from the group consisting of: 37 ℃ and 5% CO2The culture medium was changed once a day.
5. The method of claim 2, wherein the in vitro culture of bovine embryonic stem cells is performed using mitomycin-treated C57 mouse fetal fibroblasts as feeder cells.
6. The method of claim 2, wherein the cell passaging method comprises: preparing a pore plate paved with feeder cells in advance; bovine embryonic stem cells were digested with TripLE Select for 3min, and the digestion was stopped with DMEM/F12 basal medium at a passage ratio of 1: 4.
7. The method of claim 2, further comprising the step of cryopreserving the in vitro cultured and passaged bovine embryonic stem cells;
the cryopreservation method comprises the following steps: programmed cryopreservation was performed using the mFresR stem cell cryopreservation solution.
8. The method of claim 5, wherein the feeder layer cells are prepared by a method comprising: thawing primary C57 mouse fetal fibroblasts at 37 ℃ in 5% CO2Carrying out amplification culture under the condition until the cell density reaches 80-90 percent, and treating for 2.5h by using a mouse fetal fibroblast culture solution containing 12 mu g/mL mitomycin;
wherein, the culture solution of the fetal fibroblast of the C57 mouse is as follows: DMEM medium containing 10% FBS and 1% Penicilin-Streptomyces.
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