WO2019109666A1 - Procédé pour la culture de cellules issues d'urine - Google Patents

Procédé pour la culture de cellules issues d'urine Download PDF

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WO2019109666A1
WO2019109666A1 PCT/CN2018/101079 CN2018101079W WO2019109666A1 WO 2019109666 A1 WO2019109666 A1 WO 2019109666A1 CN 2018101079 W CN2018101079 W CN 2018101079W WO 2019109666 A1 WO2019109666 A1 WO 2019109666A1
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medium
urine
cells
platelet lysate
derived
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PCT/CN2018/101079
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Chinese (zh)
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王淋立
关春燕
陈月花
莫健
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皓昇莱生物制药有限公司
王淋立
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/80—Undefined extracts from animals
    • C12N2500/84—Undefined extracts from animals from mammals

Definitions

  • the invention belongs to the field of cell culture, and particularly relates to a method for culturing cells derived from urine.
  • heterologous components contained in the culture medium are at risk of introduction of the foreign virus, and in addition, the heterologous component may also become a heterologous antigen to cause an immune response, which seriously hinders the urine cell-derived cells in clinical cell therapy, Applications such as clinical regenerative medicine.
  • Urine-derived cell culture media has been continuously improved in recent years. EGF, insulin, transferrin, ethanolamine, selenium, triiodothyronine, retinoic acid, hydrocortisone, adenine, B27, etc. are constantly being tried to add urine. Substituting animal serum or other heterologous substances in the source cell culture medium, but the actual situation is that when the animal serum or other heterologous components are completely lacking, it is extremely difficult or impossible to obtain the urine we need in the presence of the above additives. Liquid source cells. During the study, it is generally found that the cell proliferation of urine-derived cells is extremely slow and cell death occurs gradually during the culture process. Therefore, it is still necessary to further explore and study to realize the culture of completely non-heterogeneous urine-derived cells.
  • Normal cells have longevity, and the number of divisions is limited. When the cells divide to a certain number of times, aging will occur. When cells other than conventional urine are used for culture, usually the sample is derived from a tissue block or an organ, and the base number is large, and the amount of cells required for the experiment can be obtained after a small number of divisions. However, the amount of urine-derived cells collected by themselves is very small, and it is necessary to carry out many divisions before it is possible to obtain the amount of cells required for research or clinical application. When the cells undergo multiple divisions, and the existing culture conditions are immature, Urine-derived cells are extremely susceptible to aging in advance.
  • cell growth generally undergoes slow growth latency, logarithmic growth phase, flattening phase, and degenerative senescence.
  • the cells can enter the logarithmic growth phase after a short latent adaptation period, and the proliferation rate is fast.
  • the amount of cells is extremely small, the cell latent growth period is prolonged, the progress of proliferation is slow, and it is difficult to enter the logarithmic growth phase, the cells are extremely difficult to survive, and apoptosis and death are highly prone to occur. Therefore, urine-derived cells are more difficult to culture in culture than other cells, and the urine-derived cells are cultured in serum-free or without any related heterologous components or extracts and obtained a research or clinically applicable amount. Technology has barely made any progress in recent decades.
  • a method for culturing a urine-derived cell wherein a urine-derived cell is cultured using a medium containing a platelet lysate.
  • the platelet lysate comprises a human platelet lysate and/or a platelet lysate of other species origin.
  • the human platelet lysate comprises a platelet lysate obtained by lysis of human blood-derived platelets, a platelet lysate lysed by platelets differentiated from various types of stem cells, and other types of cells are reprogrammed and transdifferentiated.
  • the medium can be divided into a non-heterogeneous medium or a heterologous medium.
  • the non-heterogeneous medium means a component of a non-human substance such as animal-derived, plant-derived, microbial-derived or fungal-derived in the medium. Since the medium does not contain heterologous components, there is no risk of introducing a foreign virus, and there is no risk of introducing a heterologous antigen to cause an immune response, which is beneficial to the cells of urine-derived cells in clinical cell therapy and clinical regenerative medicine. Etc. applications.
  • the platelet lysate is a component which must be added to the culture medium of the urine-derived cells of the present invention.
  • human platelet lysates include various essential factors that promote cell growth, such as transforming growth factor ⁇ 1 (TGF- ⁇ 1), transforming growth factor ⁇ 2 (TGF- ⁇ 2), and fibroblast growth factor (FGF).
  • TGF- ⁇ 1 transforming growth factor ⁇ 1
  • TGF- ⁇ 2 transforming growth factor ⁇ 2
  • FGF fibroblast growth factor
  • IGF-1 insulin-like growth factor 1
  • PDGF-AA platelet-derived growth factor AA
  • PDGF-AB platelet-derived growth factor AB
  • PDGF-BB platelet-derived growth factor BB
  • EGF epidermal growth factor
  • VEGF vascular endothelial growth factor
  • PF-4 platelet factor-4
  • albumin lipoprotein
  • adhesion factor protease inhibitor, mitogen and coagulation factors
  • mitogen and coagulation factors can greatly promote cell proliferation. Therefore, platelet lysate can be used as a substitute for fetal bovine serum to culture primary cells derived from urine, thereby solving the defect of the immune response caused by the antigen brought by the use of fetal bovine serum, and introducing the self-contained heterologous virus microorganism. And other issues.
  • Applicants have confirmed through extensive screening experiments over the years that platelet lysates have been identified as core supplements to culture media in the absence of heterologous urine-derived cells.
  • the medium of the present invention does not survive after a certain period of time in the presence of other components without the addition of platelet lysate. Under the condition that the culture medium contains platelet lysate and does not contain any of the other components, the urine-derived cells can still proliferate but the proliferation rate is affected to some extent.
  • the platelet lysate may be selected without being limited to human platelet lysate; in addition, components of the conventional urine-derived cell culture medium currently available on the market may be added to the culture medium, including A heterologous component, such as bovine serum albumin. Urine-derived cells were co-cultured with bovine serum albumin and platelet lysate.
  • the content of the platelet lysate in the medium is preferably from 0.5% to 20%, more preferably from 8% to 20% by volume.
  • the medium further contains at least one of vitamin C, a Wnt activator, and a ROCK inhibitor.
  • vitamin C a Wnt activator
  • ROCK inhibitor a ROCK inhibitor
  • vitamin C is a variety of histone demethylase cofactors, which can activate the demethylases Kdm4b, Kdm2a and so on. Kdm2a also increases cell survival, proliferation, cycle and inhibition of cellular senescence, and interacts with Oct4 to activate the pluripotent microRNA miR-302/367cluster. Vitamin C also induces the expression of core pluripotency genes and increases the level of DNA demethylation in embryonic stem cells. Therefore, vitamin C plays an important role in maintaining the ability of cells to self-renew.
  • the invention adopts vitamin C and platelet lysate to compound, vitamin C can resist the aging of cells due to the increase of cell division times, and adding vitamin C to the medium containing platelet lysate can prevent a small amount of urine-derived cells from passing through. A large number of cells divide to obtain more cells, and irreversible aging occurs. Vitamin C and platelet lysate synergistically promote urine-derived cells for effective continuous cell culture.
  • the vitamin C is preferably at least one of L-ascorbic acid, magnesium vitamin C phosphate, or the like.
  • L-ascorbic acid can promote the production of iPSCs and improve the reprogramming efficiency
  • IC 50 is 6.5 ⁇ M
  • L-ascorbic acid CAS No: 50-81-7 the structure is as follows:
  • the vitamin C is preferably L-ascorbic acid, and the content of L-ascorbic acid in the medium is preferably from 1 to 700 ⁇ g/ml, more preferably from 10 to 200 ⁇ g/ml.
  • Wnt signaling plays an important role in maintaining self-renewal of stem cells.
  • ⁇ -catenin acts as a coactivator of transcription factors and activates pluripotency-related genes such as c-myc, Oct4, Sox2, and Nanog.
  • the Wnt activator activates the Wnt signaling pathway, it can cause ⁇ -catenin to aggregate in the nucleus to maintain stem cell self-renewal ability.
  • the Wnt signaling pathway plays an important role in promoting stem cell proliferation.
  • Wnt activator can maintain cell dryness and promote cell proliferation. Adding Wnt activator to the medium containing platelet lysate can maintain the dryness of urine-derived cells during primary culture, and promote cell growth. Good proliferation.
  • the Wnt activator comprises at least one of CHIR 99021, BIO, WNT-3a, R-spondin-2.
  • CHIR 99021 is a Wnt activator with an IC 50 of 10 nM/6.7 nM and a CAS No. 252917-06-9 of CHIR 99021.
  • the structure is as follows:
  • BIO is a Wnt activator with an IC 50 of 5 nM and a BIO of CAS No: 667463-62-9.
  • the structure is as follows:
  • the Wnt activator is preferably CHIR 99021, and the content of CHIR 99021 in the medium is preferably 0.0005 to 5 ⁇ M, more preferably 0.001 to 0.5 ⁇ M.
  • Platelet lysate is compounded with ROCK inhibitor
  • Rho-related kinase plays an important role in cell function through extracellular signals, including contraction, exercise, proliferation, differentiation and apoptosis.
  • ROCK mediates vacuoles, enhances actin contraction, and activates the caspase signaling cascade and apoptosis.
  • ROCK inhibitors are small molecule inhibitors that inhibit the action of Rho-related kinases.
  • ROCK inhibitor can inhibit cell differentiation and apoptosis, improve stem cell survival and maintain its self-renewal ability.
  • the cell adherence ability can be enhanced, and the apoptosis of the cells of the urine-derived cells in the latent growth phase can be inhibited, thereby further enhancing the cell proliferation efficiency.
  • the ROCK inhibitor comprises at least one of Y-27632, Thiazovivin, Fasudil, GSK429286A, Rk1-1447.
  • Thiazovivin is a novel ROCK inhibitor with an IC 50 of 0.5 ⁇ M, which promotes the survival of stem cells.
  • Thiazovivin's CAS No: 1226056-71-8 the structure is as follows:
  • Y-27632 is an ATP-competitive ROCK-I and ROCK-II inhibitor that acts on ROCK-I and ROCK-II with Ki of 220 nM and 300 nM, respectively.
  • CAS No. 129830-38-2 of Y-27632 the structure is as follows:
  • Fasudil hydrochloride was ROCK-II, PKA, PKG, PKC and MLCK inhibitors with Ki of 0.33 ⁇ M, 1.6 ⁇ M, 1.6 ⁇ M, 3.3 ⁇ M and 36 ⁇ M, respectively.
  • Fasudil's CAS No: 105628-07-7 the structure is as follows:
  • GSK429286A is a selective ROCK1 and ROCK2 inhibitor with IC50 values of 14 nM and 63 nM.
  • CAS No. 864082-47-3 of GSK429286A has the following structure:
  • Rk1-1447 is a potent ROCK1 and ROCK2 inhibitor with IC50 values of 14.5 nM and 6.2 nM.
  • CAS No: 1342278-01-6 of Rk1-1447 the structure is as follows:
  • the ROCK inhibitor is preferably Thiazovivin, and the content of Thiazovivin in the medium is preferably 0.01 to 30 ⁇ M, more preferably 0.1 to 10 ⁇ M.
  • the medium further comprises at least one of epidermal growth factor, insulin, hydrocortisone, transferrin, epinephrine, and triiodothyronine.
  • Epidermal growth factor is an important growth factor that plays an important role in regulating cell growth, survival, migration, apoptosis, and proliferation.
  • Insulin is a protein hormone secreted by pancreatic islet ⁇ cells in the pancreas by endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, and the like.
  • endogenous or exogenous substances such as glucose, lactose, ribose, arginine, glucagon, and the like.
  • the binding of insulin to IGF-IR mediates IGF-IR autophosphorylation, which activates PI3K, which leads to increased expression of PIP3 and ultimately activates AKT, which affects cell proliferation, differentiation, apoptosis and intracellular glucose turnover.
  • inhibition of insulin function or insufficient signal will affect the ability of stem cells to self-renew, leading to cell differentiation and playing an important role in maintaining the ability of cells to self-renew.
  • Hydrocortisone is a steroid hormone produced by the adrenal cortex and has anti-inflammatory and immunosuppressive effects.
  • Iron ions can promote the rapid proliferation of cells.
  • the presence of iron ions can also affect DNA synthesis, gene regulation, etc.
  • the redox reaction of iron ions promotes the formation of highly reactive oxygen species, while high reactive oxygen species can cause oxidative stress. Peroxidation, DNA damage and ultimately cell death. Therefore, the lack or excess of intracellular iron ions can have a significant impact on cells, and transferrin, a glycoprotein, is mainly responsible for the transport of iron ions in cell culture, followed by endogenous iron ions. It plays an important role in maintaining the balance of iron.
  • Adrenaline is a hormone and neurotransmitter, and adrenergic receptor activation stimulates DNA synthesis and increases cell survival.
  • Triiodothyronine is a thyroid hormone, and triiodothyronine binds to the thyroid hormone receptor ⁇ 1 to activate the MAPK (ERK1/2) signaling pathway, thereby promoting cell proliferation.
  • the medium contains the following components: platelet lysate, vitamin C, Wnt activator, ROCK inhibitor, epidermal growth factor, insulin, hydrocortisone, transferrin, adrenaline, triiodo Adenosine, and basal medium.
  • the content of each component in the medium is: epidermal cell growth factor 1 to 100 ng/ml, insulin 1 to 75 ⁇ g/ml, hydrocortisone 1 to 360 ng/ml, transferrin 0.5 to 75 ⁇ g/ml, Epinephrine 0.1 ⁇ 5 ⁇ g/ml, triiodothyronine 0.1 ⁇ 200pg/ml, L-ascorbic acid 10-200 ⁇ g/ml, Wnt activator CHIR 99021 0.001 ⁇ 0.5 ⁇ M, ROCK inhibitor Thiazovivin 0.1 ⁇ 10 ⁇ M, platelet lysis 0.5% v/v to 20% v/v, basal medium supplemented to 1L.
  • the basal medium comprises at least one or a plurality of the above basal mediums of DMEM, DMEM/F12, ⁇ MEM, RPMI 1640, CMRL-1066, Ham's F12, IMDM, 199, MCDB mixed in any ratio.
  • the medium has no concentration requirements and is a uniform concentration of commercially available products.
  • the basal medium is mixed by DMEM/F12 or DMEM or DMEM/F12 and DMEM in any ratio.
  • human platelet lysate can be used as a core additive for culture medium without heterologous urine-derived cells, which can overcome the urine that existed in the past without heterologous culture. Source cells proliferate very slowly and gradually cause cell death and other problems.
  • a heterologous medium supplemented with human platelet lysate is capable of better culturing urine-derived cells to obtain the amount of cells required for research or clinical applications.
  • the invention uses platelet lysate for the cultivation of cells derived from urine, which can greatly promote the induction of pluripotent stem cells by urine-derived cells and promote the clinical application of pluripotent stem cells.
  • the use of the platelet lysate of the present invention in the non-heterogeneous culture and medium of urine-derived cells is innovative and has great significance for regenerative medicine.
  • the invention adds a platelet lysate to a non-heterogeneous medium of urine-derived cells, which belongs to a human-derived component and avoids the presence of xenogeneic substances.
  • the selected human platelet lysate can be obtained from healthy blood donors and detected by immunity and pathogens. .
  • human platelet lysates include various essential factors that promote cell growth, including TGF- ⁇ 1, TGF- ⁇ 2, FGF, IGF-1, PDGF-AA, PDGF-AB, PDGF-BB, EGF, VEGF, platelet factor-4 (PF-4), adhesion factors, protease inhibitors, mitogens and coagulation factors can greatly promote cell proliferation.
  • the invention adds the platelet lysate to the culture medium and has been verified by many experiments to directly separate the cells contained in the urine and expand the culture.
  • the experimental results also show that the medium of the invention greatly improves the cells of the urine source. Proliferation speed and prolonged life cycle.
  • the primary urine-derived cells are obtained by the culture medium supplemented with the platelet lysate of the present invention, and the cells are in the shape of rice granules, and the cells are observed on the 3rd to 5th day of the initial culture of the urine-derived cells. In the wall, 3 to 5 cell clones can be observed, and the cells can be passaged on the 15th day, which is better than the culture medium of 20% fetal bovine serum on urine-derived cells (R.Belik, W .Follmann, GH Degen, PH Roos, M. Blaszkewicz, HJ Knopf, K. Golka, Improvements in culturing exfoliated urothelial cells in vitro from human urine, Journal of toxicology and environmental health. Part A, 71 (2008) 923-929. ).
  • the present invention utilizes vitamin C and platelet lysate for compounding non-heterogeneous culture of cells derived from urine.
  • Vitamin C can resist the aging of cells due to the increase in the number of cell divisions. Therefore, the addition of vitamin C to the medium containing platelet lysate can prevent a small amount of urine-derived cells from undergoing a large number of cells to obtain a larger number of cells and then irreversible.
  • the aging, vitamin C and platelet lysate synergistically promote urine-derived cells for effective continuous cell culture.
  • Wnt activator can maintain cell dryness and promote cell proliferation. Adding Wnt activator to the medium containing platelet lysate can maintain the dryness of urine-derived cells during primary culture, and promote cell growth. Good proliferation.
  • the cell adherence ability can be enhanced, and the apoptosis of the cells of the urine-derived cells in the latent growth phase can be inhibited, thereby further enhancing the cell proliferation efficiency.
  • FIG. 10 Urine-derived cells collected and cultured
  • Figure 11 is a urine-derived cell culture senescence test chart.
  • the platelet lysate of the present invention which is a human-derived component, avoids the presence of xenogeneic substances, and the selected human platelet lysate is obtained from healthy blood donors and detected by immunization and pathogens.
  • the different mediums were mixed, first added to the components other than the basic medium, and then added to the basic medium for supplementation, wherein the basic medium included DMEM, DMEM/F12, ⁇ MEM, RPMI 1640 , CMRL-1066, Ham's F12, IMDM, 199, MCDB.
  • the basic medium included DMEM, DMEM/F12, ⁇ MEM, RPMI 1640 , CMRL-1066, Ham's F12, IMDM, 199, MCDB.
  • the primary urine-derived cells were digested and counted, and 1500 cells were plated per well (96-well plate).
  • Day0 was cultured using urine primary cell culture medium (REGM medium), and Day1, Day3, and Day5 were replaced with corresponding groups.
  • the medium was cultured, and 150 ⁇ l of the medium per well was subjected to MTT assay on Day 1, Day 4, and Day 7.
  • Figure 1 is the result of MTT.
  • the principle of MTT detection is that succinate dehydrogenase in living cell mitochondria can reduce exogenous MTT to water-insoluble blue-purple crystal formamidine and deposit in cells, while dead cells do not have this function.
  • DMSO can dissolve the hyperthyroidism in the cells, and the light absorption value is measured by a microplate reader.
  • the amount of MTT crystal formation is proportional to the number of cells in a certain number of cells. The number of viable cells is judged based on the measured absorbance value (OD value).
  • each group of culture medium supplemented with platelet lysate and containing no heterologous components can proliferate and culture urine-derived cells.
  • the medium solution was prepared according to Table 2, wherein the volume ratio of the platelet lysate to the medium was added in accordance with Table 3 to obtain different groups of the medium.
  • Table 2 contains different proportions of platelet lysate medium components
  • the primary urine-derived cells were digested and counted, and 1500 cells were plated per well (96-well plate).
  • Day0 was cultured using urine primary cell culture medium (REGM medium), and Day1, Day3, and Day5 were replaced with corresponding groups.
  • the medium was cultured, and 150 ⁇ l of the medium per well was subjected to MTT assay on Day 1, Day 4, and Day 7.
  • the MTT values of Groups 1-18 were as shown in Figure 2 by MTT test.
  • Fig. 2 It can be seen from Fig. 2 that different volume fractions of platelet lysate can proliferate and culture the primary urine-derived cells.
  • group 12 that is, medium containing 10% of platelet lysate, has the highest MTT value.
  • group 10-18 that is, the MTT value of the medium containing the platelet lysate volume ratio of 8% to 20% is second.
  • Platelet lysate and multivitamin C were added to the medium formulation of Table 4, and vitamin C included L-ascorbic acid, vitamin C phosphate magnesium.
  • the primary urine-derived cells were digested and counted, and 1500 cells were plated per well (96-well plate).
  • Day0 was cultured using urine primary cell culture medium (REGM medium), and Day1, Day3, and Day5 were replaced with corresponding groups.
  • the medium was cultured, 150 ⁇ l per well medium; MTT assay was performed on Day 1, Day 4, Day 7.
  • the MTT results for groups 1-7 are shown in Figure 3 by MTT test. It can be seen from Fig. 3 that in Day 7, the addition of different types and concentrations of vitamin C can promote cell proliferation in different degrees, and the MTT value of group 3 is the highest.
  • the primary urine-derived cells were digested and counted, and 1500 cells were plated per well (96-well plate).
  • Day0 was cultured using urine primary cell culture medium (REGM medium), and Day1, Day3, and Day5 were replaced with corresponding groups.
  • the medium was cultured, and 150 ⁇ l of the medium per well was subjected to MTT assay on Day 1, Day 4, and Day 7.
  • the MTT results of groups 1-9 are shown in Figure 4. Different concentrations of L-ascorbic acid can proliferate and culture the primary urine-derived cells. At Day 7, group 4, ie, the concentration of L-ascorbic acid The MTT value was the highest in the medium of 35 ⁇ g/ml. Secondly, it is group 3-7, that is, the MTT value of the medium containing L-ascorbic acid at a concentration of 10 to 200 ⁇ g/ml is relatively high. The preferred addition content of L-ascorbic acid is 10 to 200 ⁇ g/ml.
  • Wnt activators include CHIR 99021, BIO, WNT-3a, R-spondin-2.
  • the primary urine-derived cells were digested and counted, and 1500 cells were plated per well (96-well plate).
  • Day0 was cultured using urine primary cell culture medium (REGM medium), and Day1, Day3, and Day5 were replaced with corresponding groups.
  • the medium was cultured, 150 ⁇ l per well medium; MTT assay was performed on Day 1, Day 4, Day 7.
  • MTT results for groups 1-14 are shown in Figure 5 by MTT test. As can be seen from Fig. 5, when Day7 was added, different types and concentrations of Wnt activators could promote cell proliferation to different extents, and group 3 had the highest MTT value.
  • the primary urine-derived cells were digested and counted, and 1500 cells were plated per well (96-well plate).
  • Day0 was cultured using urine primary cell culture medium (REGM medium), and Day1, Day3, and Day5 were replaced with corresponding groups.
  • the medium was cultured, and 150 ⁇ l of the medium per well was subjected to MTT assay on Day 1, Day 4, and Day 7.
  • the MTT results for groups 1-9 are shown in Figure 6 by MTT test. It can be seen from Fig. 6 that different concentrations of CHIR 99021 can promote the proliferation of urine-derived cells.
  • the best group is the fifth group, that is, CHIR 99021 has the best concentration of 0.025 ⁇ M, and secondly, group 3- 8, the medium containing CHIR99021 concentration of 0.001 ⁇ 0.5 ⁇ M MTT value is relatively high. It is indicated that the preferred addition content of CHIR 99021 is 0.001 to 0.5 ⁇ M.
  • ROCK inhibitors include Y-27632, Thiazovivin, Fasudil, GSK429286A, Rk1-1447.
  • the primary urine-derived cells were digested and counted, and 1500 cells were plated per well (96-well plate).
  • Day0 was cultured using urine primary cell culture medium (REGM medium), and Day1, Day3, and Day5 were replaced with corresponding groups.
  • the medium was cultured, 150 ⁇ l per well medium; MTT assay was performed on Day 1, Day 4, Day 7.
  • the primary urine-derived cells were digested and counted, and 1500 cells were plated per well (96-well plate).
  • Day0 was cultured using urine primary cell culture medium (REGM medium), and Day1, Day3, and Day5 were replaced with corresponding groups.
  • the medium was cultured, 150 ⁇ l per well medium; MTT assay was performed on Day 1, Day 4, Day 7.
  • the MTT results for groups 1-15 are shown in Figure 8 by MTT test. It can be seen from Fig. 8 that each group of culture medium can proliferate and culture the cells from urine. At Day 7, the addition of different concentrations of thiazovivin can promote cell proliferation to different extents, and the MTT value of group 7 is the highest.
  • the primary urine-derived cells were digested and counted, and 1500 cells were plated per well (96-well plate).
  • Day0 was cultured in urine primary cell culture medium REGM medium, and Day1, Day3, and Day5 were replaced with corresponding group media.
  • the culture was carried out, and 150 ⁇ l of the medium per well; Day1, Day 4, and Day 7 were subjected to MTT assay.
  • the MTT results for groups 1-8 are shown in Figure 9 by MTT test. As can be seen from Fig. 9, each group of culture medium can proliferate and culture urine-derived cells. At Day 7, the MTT values of vitamin C, CHIR 99021, and Thiazovivin are the highest. When compounded with platelet lysate and vitamin C, platelet lysate and Wnt activator are compounded, and platelet lysate and ROCK inhibitor are compounded, these three compounding effects are equivalent when platelet lysate and vitamin C When the Wnt activator and the ROCK inhibitor are simultaneously compounded, the effect is greater than the effect of the two-two combination.
  • a primary urine-derived cell culture medium was prepared using Group 8 in Example 9. Collect 10 ml-2L of urine, centrifuge at 1010g for 5min, discard the supernatant, resuspend the pellet with PBS containing double antibody and centrifuge again to discard the supernatant, then resuspend the pellet into cell culture using urine primary cell culture medium. Plate (24-well plate), and add antibiotic primoycin (1:500), then placed in a 37 ° C, 5% CO 2 cell incubator for cultivation, without other operations, after five days, change the solution, wait for cell cloning It is grown to a certain size for digestion and passage.
  • the collected primary urine-derived cells were cultured as shown in FIG. As can be seen from the figure, the cells are in the shape of rice granules, and cell attachment can be observed on the 3rd to 5th day of urine-derived cell culture, and finally 3 to 5 cell clones can be observed, and cells can be observed on the 15th day.
  • Subculture treatment which is superior to the culture medium of 20% fetal bovine serum on urine-derived cells (R. Belik, W. Follmann, GH Degen, PH Roos, M. Blaszkewicz, HJ Knopf, K. Golka, Improvements in culturing exfoliated urothelial cells in vitro from human urine, Journal of toxicology and environmental health. Part A, 71 (2008) 923-929.). Therefore, the heterogeneous urine-derived cell culture medium obtained according to the group 8 dosing in Example 9 can be collected and cultured to obtain primary urine-derived cells.
  • the primary urine-derived cell culture medium was administered in accordance with each of the groups in Example 9.
  • the primary urine-derived cells were digested and counted, and 2000 cells were plated per well (24-well plate).
  • Day0 was cultured using urine primary cell culture medium (REGM medium), and Day1, Day3, and Day5 were replaced with corresponding groups.
  • the medium was cultured, and 500 ⁇ l of the medium was added to each well, and Day 7 was subjected to senescence detection.
  • the ⁇ -galactosidase senescence test the staining results of day 7 of groups 1-8 are shown in Fig. 11.
  • each group of culture medium can proliferate and culture the cells derived from urine, and the cells are relatively young.
  • the staining results show that the cells in the eighth group are the youngest.
  • the above experimental data shows that the medium of the present invention is in the epidermal cell growth factor of 1 to 100 ng/ml, insulin 1 to 75 ⁇ /ml, hydrocortisone 1 to 360 ng/ml, transferrin 0.5 to 75 ⁇ /ml, and epinephrine 0.1.

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Abstract

L'invention concerne un procédé pour la culture de cellules issues d'urine, qui se rapporte au domaine de la culture de cellules. Dans le procédé de culture, des cellules issues d'urine sont mises en culture à l'aide d'un milieu de culture contenant un lysat de plaquettes sanguines. Dans le criblage, il est confirmé que le lysat de plaquettes sanguines d'origine humaine servant d'additif de base du milieu de culture lorsque des cellules issues d'urine sans hétérologie sont mises en culture permet de surmonter les problèmes de très lente prolifération de cellules issues d'urine, de mort progressive de cellules et similaires dans une culture sans hétérologie selon l'état de la technique. Le milieu de culture sans hétérologie additionné du lysat de plaquettes sanguines d'origine humaine permet une meilleure culture de cellules issues d'urine et une quantité de cellules dont on a besoin dans des recherches ou des applications cliniques peut être obtenue.
PCT/CN2018/101079 2017-12-05 2018-08-17 Procédé pour la culture de cellules issues d'urine WO2019109666A1 (fr)

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CN201711267508.8 2017-12-05
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CN201810067658.2 2018-01-24
CN201810067658.2A CN108570446A (zh) 2017-12-05 2018-01-24 一种尿液来源细胞的培养方法

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EP4031151A4 (fr) * 2019-09-20 2023-10-11 Emory University Tissus de type muscle endothéliaux et lisses produits à partir de cellules urinaires et utilisations associées

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