WO2019109668A1 - 一种用于培养尿液来源细胞的培养基 - Google Patents

一种用于培养尿液来源细胞的培养基 Download PDF

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WO2019109668A1
WO2019109668A1 PCT/CN2018/101086 CN2018101086W WO2019109668A1 WO 2019109668 A1 WO2019109668 A1 WO 2019109668A1 CN 2018101086 W CN2018101086 W CN 2018101086W WO 2019109668 A1 WO2019109668 A1 WO 2019109668A1
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urine
cells
platelet lysate
derived
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王淋立
关春燕
陈月花
李强
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皓昇莱生物制药有限公司
王淋立
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Definitions

  • the invention belongs to the field of cell culture, and particularly relates to a medium 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 medium for culturing cells derived from urine the medium containing 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.
  • human platelet lysate can be used.
  • the platelet lysate is a component that must be added to the urine-derived cell culture medium 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.
  • the platelet lysate acts as a core additive to the cell culture medium without heterologous urine.
  • 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, including heterogeneity.
  • a 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 enhances cell survival, proliferation, cycle and inhibition of cellular senescence, and interacts with Oct4 to activate the pluripotent microRNA miR-302/367 cluster. 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. Irreversible senescence occurs after a large number of cells divide to obtain a larger number of cells, and 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-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.
  • An additive to a medium that is a platelet lysate is an additive to a medium that is 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.
  • human platelet lysate is the core additive of cell culture medium without heterologous urine, which can overcome the urine-derived cells that existed in the past without heterologous culture.
  • the rate of proliferation is extremely slow and problems such as cell death gradually occur.
  • the heterologous medium containing human platelet lysate is added and the urine-derived cells can be well cultured to obtain the amount of cells required for research or clinical application.
  • 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 after repeated experiments, the cells contained in the urine can be directly isolated and expanded, and the experimental results also show that the medium of the invention greatly improves the urine-derived cells. The rate of proliferation and prolonged life cycle.
  • the primary urine-derived cells were collected by the culture medium supplemented with the platelet lysate of the present invention, and the cells were in the shape of a rice-shaped shuttle, and the cells were observed to adhere to the cells on the 3rd to 5th day of the initial culture of the urine-derived cells. Finally, 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

提供了一种用于培养尿液来源细胞的培养基，所述培养基中含有人源血小板裂解物。人源血小板裂解物作为无异源性尿液来源细胞培养基的核心添加物，可克服以往无异源性培养时所存在的尿液来源细胞增殖速度极慢并且逐渐发生细胞死亡等问题。

Description

一种用于培养尿液来源细胞的培养基 技术领域
本发明属于细胞培养领域，具体涉及一种用于培养尿液来源细胞的培养基。
背景技术
近年来随着诱导多能干细胞技术的成熟，多种不同类型细胞均可成功诱导成多能干细胞，包括尿液来源细胞，据文献报道，2011年尿液来源细胞被成功诱导成为多能干细胞。而尿液来源细胞相较于其他类型的细胞提取方法更简单，只需要收集供体的尿液即可，避免对供体造成疼痛或创伤，因此尿液来源细胞是诱导多能干细胞供体的理想来源。由于尿液来源细胞的培养是获得诱导多能干细胞的关键步骤，因此，发展适合临床级别尿液来源细胞的培养技术对再生医学具有极大的推进作用。
1972年Southerland和Bain通过收集新生儿尿液并离心进而弃除上清获得少量细胞沉淀，然后用含30％胎牛血清的培养基将细胞沉淀悬浮，进而进行培养，首次从尿液中收集培养得到尿液来源细胞。之后数十年关于尿液来源细胞培养技术得到了极大的发展，然而这些用于培养尿液来源细胞的培养基都含有胎牛血清或者其它动物源性等异源性成分。除了动物性异源性成分，异源性成分还包括植物源性、微生物源性及真菌源性等非人源物质的成分。培养基中所含有的这些异源性成分存在引入外源病毒的风险，此外，异源性成分也会成为异源抗原从而引起免疫应答，这严重地阻碍了尿液来源细胞在临床细胞治疗、临床再生医学方面等的应用。
尿液来源细胞培养基近年来不断改进，EGF、胰岛素、转铁蛋白、乙醇胺、硒、三碘甲状腺氨酸、视黄酸、氢化可的松、腺嘌呤、B27等在不断被尝试加入尿液来源细胞培养基中来替代动物血清或其他异源物质，但实际情况是当完全缺乏动物血清或其它异源性成分时，在上述添加物存在下极难或不能获得我们所需应用量的尿液来源细胞。研究期间通常会发现在培养过程中尿液来源细胞增殖速度极慢并且逐渐发生细胞死亡，因此要实现完全无异源性尿液来源细胞的培养仍需要经过深入探索和研究。
有实验数据表明，从尿液中收集并成功培养到尿液来源细胞与尿液样本的体积、pH值、渗透压、尿液供体年龄、尿液中的草酸含量、尿液中淀粉酶活性及与尿液在膀胱中存留的时间有相当大的联系。从1L的尿液中仅可收集到0－6300个尿液来源细胞，其中大部分样本可收集到的尿液来源细胞量少于1000个，而可收集到尿液来源细胞并存活下来的尿液样本量仅占所有尿液样本量的37％。上述获得足够尿液来源细胞的样本用于培养时，培养条件均为含20％的胎牛血清的培养基，在不含血清的培养基中无法培养得到尿液来源细胞。即便是利用20％的胎牛血清的培养基进行尿液来源细胞培养时，收集到的大部分细胞呈现球状形态且无法进行贴壁生长，而且贴壁后的细胞大多为呈现纺锤状形态生长的单个细胞，培养一段时间后大部分只可观察到1到2个细胞增殖克隆，并且需要培养接近1个月才可增殖到可传代的细胞数量(R.Belik,W.Follmann,G.H.Degen,P.H.Roos,M.Blaszkewicz,H.J.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.)。
正常细胞是有寿命的，分裂次数有限，当细胞分裂到一定次数后会出现衰老。常规的尿液以外来源的细胞用于培养时，通常样本来源于组织块或器官，其基数较大，经过较少的分裂次数即可获得实验所需细胞量。而尿液来源细胞因本身收集到的量极少，需进行很多次分裂后才可能获得研究或临床应用所需的细胞量，当细胞经过多次分裂，加上现有的培养条件不成熟，尿液来源细胞极易提前衰老。此外，细胞生长一般会经历生长缓慢的潜伏期、对数生长期、平顶期及退化衰老期。当初始细胞量大时，因微环境的影响，细胞经过短暂的潜伏适应期后即可进入对数生长期，增殖速度快。但是当细胞量极少时，细胞潜伏生长期延长，增殖进度慢，较难进入对数生长期，细胞极难存活，极易发生凋亡和死亡等现象。因此尿液来源细胞在培养相较于其他细胞培养难度更大，要在无血清或无任何相 关的异源性成分或提取物条件下培养尿液来源细胞并获得研究或临床可应用的数量的技术在近几十年几乎无任何进展。
发明内容
本发明的目的在于提供一种用于培养尿液来源细胞的培养基(包括无异源性培养基和异源性培养基)。
本发明所采取的技术方案是：
一种用于培养尿液来源细胞的培养基，所述培养基中含有血小板裂解物。
优选的，所述血小板裂解物包括人源血小板裂解物和或/其它物种来源的血小板裂解物。
优选的，所述人源血小板裂解物包括人的血液来源的血小板裂解得到的血小板裂解物，多种类型干细胞分化而来的血小板裂解而来的血小板裂解物，其他类型细胞重编程转分化而来的血小板裂解而来的血小板裂解物，或利用细胞融合等生物学手段所获取的血小板裂解而来的血小板裂解物中的至少一种。
优选的，所述培养基可以分为无异源性培养基或异源性培养基。无异源性培养基是指的培养基中无动物源性、植物源性、微生物源性及真菌源性等非人源物质的组分。由于培养基中不含有异源性成分，所以不存在引入外源病毒的风险，也不存在引入异源抗原从而引起免疫应答的风险，有利于尿液来源细胞在临床细胞治疗、临床再生医学方面等的应用。
所以如果需要获得无异源性培养基，则选用人源血小板裂解物即可。
血小板裂解物是本发明所述尿液来源细胞培养基所必须添加组分。如同胎牛血清，人血小板裂解物因包括各种能够促进细胞生长的必需因子，如转化生长因子β1(TGF-β1)、转化生长因子β2(TGF-β2)、成纤维细胞生长因子(FGF)、胰岛素样生长因子1(IGF-1)、血小板衍生生长因子AA(PDGF-AA)、血小板衍生生长因子AB(PDGF-AB)、血小板衍生生长因子BB(PDGF-BB)、表皮细胞生长因子(EGF)、血管内皮生长因子(VEGF)、血小板因子-4(PF-4)、白蛋白、脂蛋白、附着因子、蛋白酶抑制剂、有丝分裂原和凝血因子等，能够极大地促进细胞增殖。因此血小板裂解物可作为胎牛血清替代物对尿液来源的原代细胞进行培养，从而解决因使用胎牛血清所带来的抗原引起免疫应答的缺陷，以及引入自带的异源性病毒微生物等问题。
申请人通过多年来大量地筛查实验并最终确认血小板裂解物作为无异源性尿液来源细胞培养基的核心添加物。本发明培养基在含有其他组分而没有添加血小板裂解物的条件下，细胞生存到一定时间后是不能存活下去。培养基含有血小板裂解物而不含其他组分中的任意一种的条件下，尿液来源细胞仍能够增殖但增殖速度会受到一定的影响。
如果不需要获得无异源性培养基，则选用血小板裂解物时，可以不限于人源血小板裂解物；此外还可以加入目前市面上常规的尿液来源细胞培养基的组分，包括异源性组分，如牛血清蛋白。利用牛血清蛋白和血小板裂解物复配共同培养尿液来源细胞。
优选的，所述培养基中血小板裂解物的含量优选为0.5％～20％，更优选为8％～20％，按体积百分比计。
优选的，所述培养基还含有维生素C、Wnt激活剂、ROCK抑制剂中的至少一种。发明人研究发现，血小板裂解物与维生素C、Wnt激活剂或者ROCK抑制剂进行复配，效果佳。
下面具体分析一下，血小板裂解物与维生素C、Wnt激活剂或者ROCK抑制剂的原理和复配配比。
1、血小板裂解物与维生素C复配
维生素C作用是：一方面，维生素C是多种组蛋白去甲基化酶辅因子，可以激活去甲基化酶Kdm4b、Kdm2a等。Kdm2a还可提高细胞存活、增殖、周期及抑制细胞衰老，与Oct4共同作用可激活多能性microRNA miR-302/367 cluster。维生素C还可诱导核心多能性基因的表达及提高胚胎干细胞的DNA去甲基化水平。因此，维生素C在维持细胞的自我更新能力方面发挥着重要作用。
本发明采用维生素C和血小板裂解物进行复配，维生素C可以抵抗由于细胞分裂次数的增加而导致细胞的衰老，在包含血小板裂解物的培养基中添加维生素C可以防止少量的尿液来源细胞经过大量细胞分裂得到较多数量细胞 后发生不可逆的衰老，维生素C与血小板裂解物协同促进尿液来源细胞进行有效持续地细胞培养。
优选的，维生素C优选为L-抗坏血酸、维生素C磷酸酯镁、或其类似衍生物中的至少一种。
其中，L-抗坏血酸可促进iPSCs的产生和提高重编程效率，IC 50为6.5μM，L-抗坏血酸的CAS No:50-81-7，结构如下：
Figure PCTCN2018101086-appb-000001
维生素C磷酸酯镁的CAS No:113170-55-1，结构如下：
Figure PCTCN2018101086-appb-000002
优选的，维生素C优选为L-抗坏血酸，L-抗坏血酸在所述培养基中的含量优选为1～700μg/ml，更优选为10～200μg/ml。
2、血小板裂解物与Wnt激活剂复配
Wnt信号通路在维持干细胞的自我更新方面发挥重要作用，β-catenin可以作为转录因子的共激活剂，激活c-myc、Oct4、Sox2、Nanog等多能性相关基因。当Wnt激活剂激活Wnt信号通路时，可导致β-catenin在细胞核内聚集从而维持干细胞自我更新能力。此外，Wnt信号通路还对促进干细胞的增殖起重要作用。
Wnt激活剂可以维持细胞的干性及促进细胞的增殖，在包含血小板裂解物的培养基中添加Wnt激活剂可以维持尿液来源细胞进行原代培养时大量增殖时的干性，从而促进细胞较好的增殖。
优选的，Wnt激活剂包括CHIR 99021、BIO、WNT-3a、R-spondin-2中的至少一种。
其中，CHIR 99021是Wnt激活剂，IC 50为10nM/6.7nM，CHIR 99021的CAS No:252917-06-9，结构如下：
Figure PCTCN2018101086-appb-000003
BIO是Wnt激活剂，IC 50为5nM，BIO的CAS No:667463-62-9，结构如下：
Figure PCTCN2018101086-appb-000004
优选的，Wnt激活剂优选为CHIR 99021，CHIR 99021在所述培养基中的含量优选为0.0005～5μM，更优选为0.001～0.5μM。
3、血小板裂解物与ROCK抑制剂复配
Rho相关激酶(ROCK)作为Rho下游靶点，通过胞外信号可在细胞功能上发挥重要作用，包括收缩，运动，增殖，分化和细胞凋亡。其中，ROCK介导膜泡，增强肌动蛋白的收缩，并激活胱天蛋白酶信号级联和细胞凋亡。ROCK抑制剂是可以抑制Rho相关激酶作用的小分子抑制剂。ROCK抑制剂作为Rho相关激酶的抑制剂，可抑制细胞的分化及凋亡，提高干细胞的存活及维持其自我更新能力。当在包含血小板裂解物的培养基中添加ROCK抑制剂可以提高细胞贴壁能力，抑制尿液来源细胞在潜伏生长期细胞所发生的凋亡，从而有利于进一步提高细胞的增殖效率。
优选的，ROCK抑制剂包括Y-27632、Thiazovivin、Fasudil、GSK429286A、Rk1-1447中的至少一种。
其中，Thiazovivin是新型的ROCK抑制剂，IC 50为0.5μM，可促进干细胞的存活。Thiazovivin的CAS No:1226056-71-8，结构如下：
Figure PCTCN2018101086-appb-000005
Y-27632是一种ATP竞争性的ROCK-I和ROCK-II抑制剂，作用于ROCK-I和ROCK-II，Ki分别为220nM和300nM。Y-27632的CAS No:129830-38-2，结构如下：
Figure PCTCN2018101086-appb-000006
Fasudil(HA-1077；AT-877)盐酸盐是ROCK-II，PKA，PKG，PKC和MLCK抑制剂，Ki分别为0.33μM，1.6μM，1.6μM，3.3μM和36μM。Fasudil的CAS No:105628-07-7，结构如下：
Figure PCTCN2018101086-appb-000007
GSK429286A是选择性的ROCK1和ROCK2抑制剂，IC50值为14nM和63nM。GSK429286A的CAS No:864082-47-3，结构如下：
Figure PCTCN2018101086-appb-000008
Rk1-1447是一种有效的ROCK1和ROCK2抑制剂，IC50值为14.5nM和6.2nM。Rk1-1447的CAS No:1342278-01-6，结构如下：
Figure PCTCN2018101086-appb-000009
发明人研发中尝试加入ROCK抑制剂如Thiazovivin、Y-27632、Fasudil、GSK429286A、Rk1-1447等并通过多次实验，其中Thiazovivin与血小板裂解物复配效果最佳，其MTT结果值显示当加入Thiazovivin时，Day7增殖数据为0.89，而其他ROCK抑制剂较这个值小，只有0.85或更小。发明人最终确定将Thiazovivin与血小板裂解物复配，作为尿液来源细胞无异源性培养基的成分。
优选的，ROCK抑制剂优选为Thiazovivin，Thiazovivin在所述培养基中的含量优选为0.01～30μM，更优选为0.1～10μM。
优选的，所述培养基还含有表皮细胞生长因子、胰岛素、氢化可的松、转铁蛋白、肾上腺素、三碘甲腺原氨酸中的至少一种。
表皮细胞生长因子是一种重要的生长因子，在调节细胞生长，存活，迁移，凋亡，增殖等方面发挥重要作用。
胰岛素是由胰脏内的胰岛β细胞受内源性或外源性物质如葡萄糖、乳糖、核糖、精氨酸、胰高血糖素等的刺激而分泌的一种蛋白质激素。一方面，胰岛素与IGF-IR结合介导IGF-IR自身磷酸化，可激活PI3K，进而导致PIP3表达提高，最终激活AKT，从而影响细胞增殖，分化，凋亡及细胞内葡萄糖运转。另一方面，胰岛素功能被抑制或 信号不足时将影响干细胞的自我更新能力，导致细胞分化，在维持细胞的自我更新能力方面发挥着重要作用。
氢化可的松是肾上腺皮质产生的类固醇激素，具有抗炎和免疫抑制作用。
铁离子可促进细胞的快速增殖，铁离子的存在还会影响DNA合成，基因调节等，此外，铁离子的氧化还原反应会促进高活性氧的形成，而高活性氧可引起氧化应激，脂质过氧化，DNA损伤并最终导致细胞死亡。因此，细胞内铁离子的缺乏或过多均可对细胞造成重大影响，而转铁蛋白，是一种糖蛋白，在细胞培养中主要是负责铁离子的运输，其次是结合内源性铁离子，对维持铁的平衡发挥重要的作用。
肾上腺素是一种激素和神经递质，肾上腺素受体激活可刺激DNA合成及提高细胞的存活。
三碘甲状腺原氨酸是一种甲状腺激素，三碘甲状腺原氨酸与甲状腺激素受体β1结合激活MAPK(ERK1/2)信号通路，从而促进细胞增殖。
优选的，所述培养基含有下述组分：血小板裂解物、维生素C、Wnt激活剂、ROCK抑制剂、表皮细胞生长因子、胰岛素、氢化可的松、转铁蛋白、肾上腺素、三碘甲腺原氨酸，及基础培养基。
优选的，所述培养基中各组分含量为：表皮细胞生长因子1～100ng/ml、胰岛素1～75μg/ml、氢化可的松1～360ng/ml、转铁蛋白0.5～75μg/ml、肾上腺素0.1～5μg/ml、三碘甲腺原氨酸0.1～200pg/ml、L-抗坏血酸10～200μg/ml、Wnt激活剂CHIR 99021 0.001～0.5μM、ROCK抑制剂Thiazovivin 0.1～10μM、血小板裂解物0.5％v/v-20％v/v、基础培养基补足至1L。
优选的，基础培养基包括DMEM、DMEM/F12、αMEM、RPMI 1640、CMRL-1066、Ham's F12、IMDM、199、MCDB中的至少一种或多种上述基础培养基以任意比例混合。所述培养基没有浓度要求，均是市售产品的统一浓度。
优选的，基础培养基是由DMEM/F12或DMEM或DMEM/F12和DMEM按照任意比例混合。
一种培养基的添加物，所述添加物为血小板裂解物。
优选的，所述血小板裂解物包括人源血小板裂解物和或/其它物种来源的血小板裂解物。
优选的，所述人源血小板裂解物包括人的血液来源的血小板裂解得到的血小板裂解物，多种类型干细胞分化而来的血小板裂解而来的血小板裂解物，其他类型细胞重编程转分化而来的血小板裂解而来的血小板裂解物，或细胞融合等生物学手段所获取的血小板裂解而来的血小板裂解物中的至少一种。
血小板裂解物在尿液来源细胞培养中的应用。
血小板裂解物在尿液来源细胞的无异源性培养中的应用。
一种尿液来源细胞，所述细胞是利用上述任一项所述培养基培养得到。
本发明的有益效果是：
申请人通过多年来大量地筛查实验，最终确认人源血小板裂解物作为无异源性尿液来源细胞培养基的核心添加物，可克服以往无异源性培养时所存在的尿液来源细胞增殖速度极慢并且逐渐发生细胞死亡等问题。添加人源血小板裂解物的无异源性培养基并能够较好地培养尿液来源细胞，获得研究或临床应用所需的细胞量。
本发明将血小板裂解物用于尿液来源细胞的培养，可极大地促进尿液来源细胞诱导得到多能干细胞并促进多能干细胞的临床应用。本发明血小板裂解物在尿液来源细胞的无异源性培养及培养基中的应用具有创新性，对再生医学具有重大的意义。
本发明在尿液来源细胞的无异源性培养基中加入血小板裂解物，它属于人来源成分，避免异种物质存在，所选用的人血小板裂解物可以从健康献血者获得并通过免疫及病原体检测。如同胎牛血清，人血小板裂解物包括各种能够促进细胞生长的必需因子，它含有TGF-β1、TGF-β2、FGF、IGF-1、PDGF-AA、PDGF-AB、PDGF-BB、EGF、VEGF、血小板因子-4(PF-4)、附着因子、蛋白酶抑制剂、有丝分裂原和凝血因子，能够极大地促进细胞增殖。
本发明在培养基中加入血小板裂解物后并经过多次实验验证可直接分离获得尿液所包含的细胞并进行扩大培养，实验结果也显示了本发明培养基极大的提高了尿液来源细胞的增殖速度及延长了生命周期。
利用本发明添加了血小板裂解物的培养基培养收集得到原代尿液来源细胞，所述细胞呈米粒状梭长形，在尿液来源细胞初始培养的第3至5天可观察到细胞贴壁，最终可观察到3至5处细胞克隆，在第15天可对细胞进行传代处理，其效果优于20％胎牛血清的培养基对尿液来源细胞的培养效果(R.Belik,W.Follmann,G.H.Degen,P.H.Roos,M.Blaszkewicz,H.J.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.)。
本发明采用维生素C和血小板裂解物进行复配用于尿液来源细胞的无异源性培养。维生素C可以抵抗由于细胞分裂次数的增加而导致细胞的衰老，因此，在包含血小板裂解物的培养基中添加维生素C可以防止少量的尿液来源细胞经过大量细胞分裂得到较多数量细胞后发生不可逆的衰老，维生素C与血小板裂解物协同促进尿液来源细胞进行有效持续地细胞培养。
Wnt激活剂可以维持细胞的干性及促进细胞的增殖，在包含血小板裂解物的培养基中添加Wnt激活剂可以维持尿液来源细胞进行原代培养时大量增殖时的干性，从而促进细胞较好的增殖。
当在包含血小板裂解物的培养基中添加ROCK抑制剂可以提高细胞贴壁能力，抑制尿液来源细胞在潜伏生长期细胞所发生的凋亡，从而有利于进一步提高细胞的增殖效率。
附图说明
图1 MTT结果；
图2 MTT结果；
图3 MTT结果；
图4 MTT结果；
图5 MTT结果；
图6 MTT结果；
图7 MTT结果；
图8 MTT结果；
图9 MTT结果；
图10尿液来源细胞收集培养形态图；
图11尿液来源细胞培养衰老检测图。
具体实施方式
本发明实施例中的血小板裂解物，它属于人来源成分，避免异种物质存在，所选用的人血小板裂解物从健康献血者获得并通过免疫及病原体检测。
下面结合实施例对本发明做进一步的说明，但不限于此。
实施例1
按照表1进行不同组别培养基的配液，先加入基础培养基以外的各组分，然后再加入基础培养基进行补齐，其中，基础培养基包括DMEM、DMEM/F12、αMEM、RPMI 1640、CMRL-1066、Ham's F12、IMDM、199、MCDB。
表1 无异源性尿液来源细胞培养基配方
Figure PCTCN2018101086-appb-000010
Figure PCTCN2018101086-appb-000011
Figure PCTCN2018101086-appb-000012
消化原代尿液来源细胞并进行计数，每孔(96孔板)铺板1500个细胞，Day0使用尿液原代细胞培养基(REGM培养基)进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔培养基150μl，于Day1，Day4，Day7进行MTT检测。
经MTT检验，第1-25组的MTT结果如图1所示。
图1是MTT的结果图，MTT检测原理为活细胞线粒体中的琥珀酸脱氢酶能使外源性MTT还原为水不溶性的蓝紫色结晶甲臢并沉积在细胞中，而死细胞无此功能。DMSO能溶解细胞中的甲臢，用酶标仪测定其光吸收值，在一定细胞数范围内，MTT结晶形成的量与细胞数成正比。根据测得的吸光度值(OD值)，来判断活细胞数量。
从图1可知，添加了血小板裂解物且不含异源性成分的各组培养基均能对尿液来源细胞进行增殖培养，在Day7时，组别1的MTT值最高，即基础培养基为DMEM：DMEM/F12＝1:1较其它基础培养基培养效果好。
实施例2
本实施例是在实施例1组别1的基础上，按照表2进行培养基配液，其中，血小板裂解物占培养基的体积比例按照表3进行添加，得到不同组别的培养基。配置时，先加入基础培养基以外的各组分，然后再加入基础培养基进行补齐，其中，基础培养基为DMEM/F12:DMEM＝1:1。
表2 含不同体积比例血小板裂解物培养基组分
Figure PCTCN2018101086-appb-000013
表3 血小板裂解物体积比例
组别 血小板裂解物(％)
1 0
2 0.5
3 1
4 2
5 3
6 4
7 5
8 6
9 7
10 8
11 9
12 10
13 11
14 12
15 14
16 16
17 18
18 20
消化原代尿液来源细胞并进行计数，每孔(96孔板)铺板1500个细胞，Day0使用尿液原代细胞培养基(REGM培养基)进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔培养基为150μl，于Day1，Day4，Day7进行MTT检测。
经MTT检验，第1-18组的MTT值如图2所示。
从图2可知，不同体积比例血小板裂解物均能对原代尿液来源细胞进行增殖培养，在Day7时，组别12，即含血小板裂解物体积比例为10％的培养基MTT值最高。其次，是组别10-18，即含血小板裂解物体积比例为8％～20％的培养基MTT值次之。
实施例3
按照表4进行不同组别培养基的配液，先加入基础培养基以外的各组分，然后再加入基础培养基进行补齐，其中，基础培养基为DMEM/F12:DMEM＝1:1。
表4的培养基配方中添加了血小板裂解物和多种维生素C，维生素C包括L-抗坏血酸、维生素C磷酸酯镁。
表4 无异源性尿液来源细胞培养基配方
Figure PCTCN2018101086-appb-000014
Figure PCTCN2018101086-appb-000015
消化原代尿液来源细胞并进行计数，每孔(96孔板)铺板1500个细胞，Day0使用尿液原代细胞培养基(REGM培养基)进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔培养基150μl；于Day1，Day4，Day7进行MTT检测。
经MTT检验，第1-7组的MTT结果如图3所示。从图3可知，在Day7时，加入不同种类及不同浓度的维生素C能在不同程度促进细胞增殖，其中组别3的MTT值最高。
实施例4
按照表5进行不同组别培养基的配液，先加入基础培养基以外的各组分，然后再加入基础培养基进行补齐，其中，基础培养基为DMEM/F12:DMEM＝1:1。
表5 无异源性尿液来源细胞培养基配方
Figure PCTCN2018101086-appb-000016
Figure PCTCN2018101086-appb-000017
消化原代尿液来源细胞并进行计数，每孔(96孔板)铺板1500个细胞，Day0使用尿液原代细胞培养基(REGM培养基)进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔培养基150μl，于Day1，Day4，Day7进行MTT检测。
经MTT检验，第1-9组的MTT结果如图4所示，不同浓度L-抗坏血酸均能对原代尿液来源细胞进行增殖培养，在Day7时，组别4，即含L-抗坏血酸浓度为35μg/ml的培养基MTT值最高。其次，是组别3-7，即含L-抗坏血酸浓度为10～200μg/ml的培养基MTT值相对较高。说明L-抗坏血酸的优选添加含量为10～200μg/ml。
实施例5
按照表6进行不同组别培养基的配液，先加入基础培养基以外的各组分，然后再加入基础培养基进行补齐，其中，基础培养基为DMEM/F12:DMEM＝1:1。
表6的培养基配方中添加了血小板裂解物和多种Wnt激活剂，Wnt激活剂包括CHIR 99021、BIO、WNT-3a、R-spondin-2。
表6 无异源性尿液来源细胞培养基配方
Figure PCTCN2018101086-appb-000018
Figure PCTCN2018101086-appb-000019
消化原代尿液来源细胞并进行计数，每孔(96孔板)铺板1500个细胞，Day0使用尿液原代细胞培养基(REGM 培养基)进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔培养基150μl；于Day1，Day4，Day7进行MTT检测。
经MTT检验，第1-14组的MTT结果如图5所示。从图5可知，在Day7时，加入不同种类及不同浓度的Wnt激活剂能在不同程度促进细胞增殖，其中组别3的MTT值最高。
实施例6
按照表7进行不同组别培养基的配液，先加入基础培养基以外的各组分，然后再加入基础培养基进行补齐，其中，基础培养基为DMEM/F12:DMEM＝1:1。
表7 无异源性尿液来源细胞培养基配方
Figure PCTCN2018101086-appb-000020
消化原代尿液来源细胞并进行计数，每孔(96孔板)铺板1500个细胞，Day0使用尿液原代细胞培养基(REGM培养基)进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔培养基150μl，于Day1，Day4，Day7进行MTT检测。
经MTT检验，第1-9组的MTT结果如图6所示。从图6可知，不同浓度的CHIR 99021均可以促进尿液来源细胞的增殖，其中最佳的组别为第5组，即CHIR 99021浓度为0.025μM时效果最好，其次，是组别3-8，即含CHIR99021浓度为0.001～0.5μM的培养基MTT值相对较高。说明CHIR 99021的优选添加含量为0.001～0.5μM。
实施例7
按照表8进行不同组别培养基配液，先加入基础培养基以外的各组分，然后再加入基础培养基进行补齐，其中，基础培养基为DMEM/F12:DMEM＝1:1。
表8的培养基配方中添加了血小板裂解物和多种ROCK抑制剂。ROCK抑制剂包括Y-27632、Thiazovivin、Fasudil、 GSK429286A、Rk1-1447。
表8 无异源性尿液来源细胞培养基配方
Figure PCTCN2018101086-appb-000021
消化原代尿液来源细胞并进行计数，每孔(96孔板)铺板1500个细胞，Day0使用尿液原代细胞培养基(REGM培养基)进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔培养基150μl；于Day1，Day4，Day7进行MTT检测。
经MTT检验，第1-17组的MTT结果如图7所示。从图7可知，在Day7时，加入不同种类及不同浓度的ROCK抑制剂能在不同程度促进细胞增殖，其中组别6的MTT值最高。
实施例8
按照表9及表10进行不同组别培养基配液，先加入基础培养基以外各组分，然后再加入基础培养基进行补齐，其中，基础培养基为DMEM/F12:DMEM＝1:1。
表9 无异源性尿液来源细胞培养基部分成分
Figure PCTCN2018101086-appb-000022
Figure PCTCN2018101086-appb-000023
表10 thiazovivin不同浓度
Figure PCTCN2018101086-appb-000024
消化原代尿液来源细胞并进行计数，每孔(96孔板)铺板1500个细胞，Day0使用尿液原代细胞培养基(REGM培养基)进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔培养基150μl；于Day1，Day4，Day7进行MTT检测。
经MTT检验，第1-15组的MTT结果如图8所示。从图8可知，各组培养基均能对尿液来源细胞进行增殖培养，在Day7时，加入不同浓度的thiazovivin能在不同程度促进细胞的增殖，其中组别7的MTT值最高。
实施例9
按照表11进行不同组别培养基配液，先加入基础培养基以外的各组分，然后再加入基础培养基进行补齐，其中，基础培养基为DMEM/F12:DMEM＝1:1。
表11 无异源性尿液来源细胞培养基配方
Figure PCTCN2018101086-appb-000025
消化原代尿液来源细胞并进行计数，每孔(96孔板)铺板1500个细胞，Day0使用尿液原代细胞培养基REGM培养基进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔培养基150μl；Day1，Day4，Day7进行MTT检测。
经MTT检验，第1-8组的MTT结果如图9所示。从图9可知，各组培养基均能对尿液来源细胞进行增殖培养，在Day7时，共同加入维生素C、CHIR 99021、Thiazovivin的组别的MTT值最高。采用血小板裂解物和维生素C进行复配，血小板裂解物和Wnt激活剂进行复配，及血小板裂解物和ROCK抑制剂进行复配时，这三种复配效果相当，当血小板裂解物与维生素C、Wnt激活剂、ROCK抑制剂这三者同时进行复配时，其效果大于其两两复配的效果。
实施例10
本实施例是利用实施例9中组别8进行原代尿液来源细胞培养基配液。收集10ml-2L的尿液，1010g离心5min，弃上清，用含双抗的PBS对沉淀进行重悬并再次进行离心弃上清，然后使用尿液原代细胞培养基重悬沉淀至细胞培养板(24孔板)，并加入抗生素primoycin(1:500)，然后放置于37℃，5％CO 2细胞培养箱内进行培养，期间不对其进行其他操作，五天后进行换液，待细胞克隆长至一定大小进行消化传代。
培养收集到的原代尿液来源细胞如图10所示。从图中可知，细胞呈米粒状梭长形，在尿液来源细胞培养的第3至5天可观察到细胞贴壁，最终可观察到3至5处细胞克隆，在第15天可对细胞进行传代处理，其效果优于20％胎牛血清的培养基对尿液来源细胞的培养效果(R.Belik,W.Follmann,G.H.Degen,P.H.Roos,M.Blaszkewicz,H.J.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.)。因此，按照实施例9中组别8配液所得到的无异源性尿液来源细胞培养基可以收集并培养得到原代尿液来源细胞。
实施例11
本实施例是按照实施例9中各组别进行原代尿液来源细胞培养基配液。消化原代尿液来源细胞并进行计数，每孔(24孔板)铺板2000个细胞，Day0使用尿液原代细胞培养基(REGM培养基)进行培养，Day1，Day3，Day5更换为对应组别培养基进行培养，每孔添加培养基500μl，Day7进行衰老检测。
进行衰老检测时，先加入PBS进行洗涤，固定完毕后再次用PBS进行洗涤，然后加入新鲜配制的β-半乳糖甘酶染色液进行37℃孵育过夜，第二天显微镜下观察染色状态。
经β-半乳糖苷酶衰老检验，第1-8组的day7的染色结果如图11所示。从图11可知，各组培养基均能对尿液来源细胞进行增殖培养，且细胞均较为年轻，其中，染色结果显示第8组的细胞最年轻。以上实验数据表明：本发明的培养基在表皮细胞生长因子1～100ng/ml、胰岛素1～75μ/ml、氢化可的松1～360ng/ml、转铁蛋白0.5～75μ/ml、肾上腺素0.1～5μg/ml、三碘甲腺原氨酸0.1～200pg/ml、维生素C 10～200μg/ml、Wnt激活剂CHIR 99021 0.001～0.5μM、ROCK抑制剂Thiazovivin 0.1～10μM，血小板裂解物0.5％v/v～20％v/v的浓度范围内，均可以促进尿液来源细胞的增殖速度及单细胞存活率。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (15)

  1. 一种用于培养尿液来源细胞的培养基，其特征在于：所述培养基中含有血小板裂解物。
  2. 根据权利要求1所述的培养基，其特征在于：所述血小板裂解物包括人源血小板裂解物和或/其它物种来源的血小板裂解物。
  3. 根据权利要求2所述的培养基，其特征在于：所述人源血小板裂解物包括人的血液来源的血小板裂解得到的血小板裂解物，多种类型干细胞分化而来的血小板裂解而来的血小板裂解物，其他类型细胞重编程转分化而来的血小板裂解而来的血小板裂解物，或利用生物学手段所获取的血小板裂解而来的血小板裂解物中的至少一种。
  4. 根据权利要求3所述的培养基，其特征在于：所述培养基中血小板裂解物的含量优选为0.5％～20％，按体积百分比计。
  5. 根据权利要求4所述的培养基，其特征在于：所述培养基还含有维生素C、Wnt激活剂、ROCK抑制剂中的至少一种。
  6. 根据权利要求5所述的培养基，其特征在于：维生素C包括L-抗坏血酸、维生素C磷酸酯镁、或其类似衍生物中的至少一种。
  7. 根据权利要求6所述的培养基，其特征在于：维生素C优选为L-抗坏血酸，L-抗坏血酸在所述培养基中的含量优选为1～700μg/ml。
  8. 根据权利要求5所述的培养基，其特征在于：Wnt激活剂包括CHIR 99021、BIO、WNT-3a、R-spondin-2中的至少一种。
  9. 根据权利要求8所述的培养基，其特征在于：Wnt激活剂优选为CHIR 99021，CHIR 99021在所述培养基中的含量优选为0.0005～5μM。
  10. 根据权利要求5所述的培养基，其特征在于：ROCK抑制剂包括Y-27632、Thiazovivin、Fasudil、GSK429286A、Rk1-1447中的至少一种。
  11. 根据权利要求10所述的培养基，其特征在于：ROCK抑制剂优选为Thiazovivin，Thiazovivin在所述培养基中的含量优选为0.01～30μM。
  12. 根据权利要求1-11任一项所述的培养基，其特征在于：所述培养基还含有表皮细胞生长因子、胰岛素、氢化可的松、转铁蛋白、肾上腺素、三碘甲腺原氨酸中的至少一种。
  13. 根据权利要求12任一项所述的培养基，其特征在于：所述培养基含有下述组分：血小板裂解物、维生素C、Wnt激活剂、ROCK抑制剂、表皮细胞生长因子、胰岛素、氢化可的松、转铁蛋白、肾上腺素、三碘甲腺原氨酸，及基础培养基。
  14. 根据权利要求13任一项所述的培养基，其特征在于：所述培养基中各组分含量为：表皮细胞生长因子1～100ng/ml、胰岛素1～75μg/ml、氢化可的松1～360ng/ml、转铁蛋白0.5～75μg/ml、肾上腺素0.1～5μg/ml、三碘甲腺原氨酸0.1～200pg/ml、L-抗坏血酸10～200μg/ml、Wnt激活剂CHIR 99021 0.001～0.5μM、ROCK抑制剂Thiazovivin0.1～10μM，血小板裂解物0.5％v/v～20％v/v，基础培养基补足至1L。
  15. 一种尿液来源细胞，其特征在于：所述细胞是利用权利要求1-14任一项所述培养基培养得到。
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