CN108570446A - A kind of cultural method of urine derived cell - Google Patents
A kind of cultural method of urine derived cell Download PDFInfo
- Publication number
- CN108570446A CN108570446A CN201810067658.2A CN201810067658A CN108570446A CN 108570446 A CN108570446 A CN 108570446A CN 201810067658 A CN201810067658 A CN 201810067658A CN 108570446 A CN108570446 A CN 108570446A
- Authority
- CN
- China
- Prior art keywords
- cell
- culture medium
- platelet
- culture
- urine
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
Landscapes
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of cultural methods of urine derived cell, belong to field of cell culture.The cultural method is cultivated urine derived cell using the culture medium comprising platelet cracking content.Applicant is by the way that screening is tested in large quantities for many years, the core additive of culture medium when final confirmation people's source platelet cracking content is as no heterologous urine derived cell culture, existing urine derived cell growth rate is extremely slow when can overcome in the past without heterologous culture and the problems such as cell death gradually occurs.Addition people source platelet cracking content without heterologous culture medium and can preferably cultivate urine derived cell, studied or clinical application needed for cell concentration.Platelet cracking content of the present invention has novelty in urine derived cell without the application in heterologous culture and culture medium, is of great significance to regenerative medicine.
Description
Technical field
The invention belongs to field of cell culture, and in particular to a kind of cultural method of urine derived cell.
Background technology
Recently as the maturation of induced multi-potent stem cell technology, a variety of different type cells can successfully induce into multipotency
Stem cell, including urine derived cell, according to the literature, urine derived cell in 2011 becomes multipotency by successfully induction to be done carefully
Born of the same parents.And urine derived cell is simpler compared to other kinds of cell extraction method, it is only necessary to the urine of donor is collected,
It avoids causing pain or wound to donor, therefore urine derived cell is the ideal source of induced multi-potent stem cell donor.Due to
The culture of urine derived cell is to obtain the committed step of induced multi-potent stem cell, therefore, develops and clinical rank urine is suitble to
The culture technique of source cell has great progradation to regenerative medicine.
Reject supernatants by collecting neonatal urine and centrifuging of Southerland and Bain in 1972 obtain a small amount of thin
Born of the same parents are precipitated, and then cell precipitation is suspended with the culture medium containing 30% fetal calf serum, and then is cultivated, is received from urine for the first time
Collection culture obtains urine derived cell.Many decades have obtained great development about urine derived cell culture technique later, so
And these are used to cultivate culture mediums of urine derived cell all containing fetal calf serum or other animal derived equal heterologous elements.
In addition to animality heterologous element, heterologous element further includes the inhuman source object such as plant-derived, microbial origin and fungal source
The ingredient of matter.There is the risk for introducing exogenous virus in these heterologous elements contained in culture medium, in addition, heterologous element
Also can become heterologous antigen so as to cause immune response, this seriously hinder urine derived cell in clinical cytology treatment, face
Application in terms of bed regenerative medicine etc..
Urine derived cell culture medium is continuously improved in recent years, EGF, insulin, transferrins, ethanol amine, selenium, triiodo first
Shape gland propylhomoserin, retinoic acid, hydrocortisone, adenine, B27 etc. are constantly being attempted addition urine derived cell culture medium
Substitute animal blood serum or other xenobiotics, but actual conditions be when complete lack of animal blood serum or other heterologous elements,
The urine derived cell extremely difficult or that our required application amounts cannot be obtained in the presence of above-mentioned additive.It would generally be sent out during research
Urine derived cell growth rate is extremely slow in present incubation and cell death gradually occurs, therefore to realize and be no different completely
There is still a need for by further investigation and research for the culture of source property urine derived cell.
Have experimental data show from urine collect and successfully cultivate to the volume of urine derived cell and urine specimen,
PH value, urine donor age, the oxalic acid content in urine, amylase activity and is deposited with urine in bladder osmotic pressure in urine
There is sizable contact in the time stayed.It is only collected from the urine of 1L to 0-6300 urine derived cell, wherein most
The urine derived cell amount that sample collects is less than 1000, and the urine for collecting urine derived cell and surviving
Sample size only accounts for the 37% of all urine specimen amounts.The sample of the above-mentioned enough urine derived cells of acquisition is for when cultivating, cultivating
Condition is the culture medium containing 20% fetal calf serum, can not cultivate to obtain urine source in the culture medium without serum thin
Born of the same parents.When even carrying out urine derived cell culture using the culture medium of 20% fetal calf serum, the most cells that are collected into
Spherical-like morphology is presented and can not carry out adherent growth, and it is adherent after cell be mostly the growth of fusiform form is presented single
Cell, 1 to 2 cell Proliferations clones only can be observed in major part after cultivating a period of time, and need culture close to 1 month
Can be proliferated can pass on cell quantity (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.)。
Normal cell has the service life, and division number is limited, will appear aging after cell division to certain number.It is conventional
Urine with the cell of external source for cultivate when, for usual samples sources in tissue block or organ, radix is larger, and process is less
Division number can be obtained experiment needed for cell concentration.And the amount that urine derived cell is collected by itself is few, need to carry out very
Repeatedly be likely to study after division or clinical application needed for cell concentration, when cell is divided by multiple, in addition existing
Condition of culture is immature, and urine derived cell easily shifts to an earlier date aging.In addition, cell growth can generally undergo slow-growing hide
Phase, exponential phase, flat-top phase and degeneration declining period.When initial cell volume is big, because of the influence of microenvironment, cell is by of short duration
The latent laundering period after can enter exponential phase, growth rate is fast.But when cell concentration is few, cell latent growth period
Extend, proliferation progress is slow, it is more difficult to enter exponential phase, the extremely difficult survival of cell, easily occur apoptosis and it is dead phenomena such as.Therefore
Urine derived cell in culture compared to other cell culture difficulty biggers, serum-free or without any relevant heterologous at
Point or extract under the conditions of culture urine derived cell and obtain the technology of research or clinical applicable quantity in recent decades
Almost without any progress.
Invention content
The purpose of the present invention is to provide a kind of cultural methods of urine derived cell (including without heterologous culture and heterologous
Property culture).
The technical solution used in the present invention is：
A kind of cultural method of urine derived cell, using the culture medium comprising platelet cracking content to urine derived cell
It is cultivated.
Preferably, the platelet cracking content includes the blood platelet of people source platelet cracking content and/or/other source of species
Lysate.
Preferably, people source platelet cracking content includes that the obtained blood platelet of platelet lysates of the blood sources of people is split
Object is solved, platelet lysates that multiple types stem cell differentiates and the platelet cracking content come, the reprogramming of other types cell
Transdifferentiation and the platelet lysates come and the platelet cracking content come, or utilize the blood acquired in the biological means such as cell fusion
At least one of the platelet cracking content of platelet cracking.
Preferably, the culture medium can be divided into no heterologous culture medium or heterologous culture medium.Without heterologous culture medium
The component of the inhuman source substance such as non-animal derived property, plant-derived, microbial origin and fungal source in the culture medium for referring to.Due to
In culture medium do not contain heterologous element, so there is no introduce exogenous virus risk, also be not present introduce heterologous antigen from
And cause the risk of immune response, be conducive to urine derived cell answering in terms of clinical cytology treatment, clinical regenerative medicine etc.
With.
So when if necessary to carry out without heterologous culture, then people source platelet cracking content is selected.
The component that culture medium must add when platelet cracking content is urine derived cell culture of the present invention.As
Fetal calf serum, human blood platelets lysate is because including the various necessary factors that can promote cell growth, such as transforminggrowthfactor-β1
(TGF-β 1), transforming grouth factor beta 2 (TGF-β 2), fibroblast growth factor (FGF), type-1 insulin like growth factor
(IGF-1), Platelet-derived growth factor A A (PDGF-AA), Platelet-derived growth factor A B (PDGF-AB), blood platelet spread out
Raw growth factor B B (PDGF-BB), epithelical cell growth factor (EGF), vascular endothelial growth factor (VEGF), blood platelet because
Son -4 (PF-4), albumin, lipoprotein, attachment element, protease inhibitors, mitogen and coagulation factor etc., Neng Gouji
The earth promotes cell Proliferation.Therefore platelet cracking content can be used as fetal bovine serum replacement and be carried out to the primary cell in urine source
Culture to solve because using antigen caused by fetal calf serum to cause the defect of immune response, and introduces included heterologous
The problems such as property viral micro-organisms.
Applicant is final to confirm that platelet cracking content is come as no heterologous urine by the way that screening is tested in large quantities for many years
The core additive of culture medium when source cell culture.Culture medium of the present invention containing other components without add platelet lysates
Under conditions of object, cells survival is to being that cannot survive down after a certain period of time.Culture medium contains platelet cracking content and is free of it
Under conditions of any one in his component, urine derived cell still is able to proliferation but growth rate can be affected to some extent.
If you do not need to carrying out without heterologous culture, then when selecting platelet cracking content, people source blood platelet being not limited to
Lysate；The component of current conventional urine derived cell culture medium on the market can additionally be added in the medium, including
Heterologous component, such as bovine serum albumin.Co-incubation urine derived cell is compounded using bovine serum albumin and platelet cracking content.
Preferably, the content of platelet cracking content is preferably 0.5%~20% in the culture medium, more preferably 8%~
20%, by volume percentage.
Preferably, the culture medium also contains at least one of vitamin C, Wnt activator, ROCK inhibitor.Invention
For people the study found that platelet cracking content is compounded with vitamin C, Wnt activator or ROCK inhibitor, effect is good.
It makes a concrete analysis of below, the principle of platelet cracking content and vitamin C, Wnt activator or ROCK inhibitor
It is matched with compounding.
1, platelet cracking content and vitamin C compound
Vitamin C acts on：On the one hand, vitamin C is a variety of histone demethylase co-factors, can activate first
Base enzyme Kdm4b, Kdm2a etc..Kdm2a also can be improved cell survival, proliferation, period and inhibit cell ageing, common with Oct4
Effect can activate versatility microRNA miR-302/367 cluster.Vitamin C also can induce core versatility gene
Expression and the DNA demethylations for improving embryonic stem cell are horizontal.Therefore, vitamin C is in the self-renewal capacity side for maintaining cell
Face plays an important role.
The present invention is compounded using vitamin C and platelet cracking content, and vitamin C can be resisted due to cell division time
Several increase and the aging for leading to cell, in the culture medium comprising platelet cracking content adding vitamin C can prevent on a small quantity
Urine derived cell irreversible aging, vitamin C and blood occurs after a large amount of cell divisions obtain a greater number cell
Platelet lysate synergistic effect promotes urine derived cell to carry out effectively constantly cell culture.
Preferably, vitamin C be preferably in L-AA, L-Ascorbic Acid L-O-Phosphate or its like derivatives at least
It is a kind of.
Wherein, L-AA can promote the generation of iPSCs and improve reprogramming efficiency, IC50It is 6.5 μM, L- Vitamin Cs
The CAS No of acid:50-81-7, structure are as follows：
The CAS No of L-Ascorbic Acid L-O-Phosphate:113170-55-1, structure are as follows：
Preferably, vitamin C is preferably L-AA, and content of the L-AA in the culture medium is preferably 1
~700 μ g/ml, more preferably 10~200 μ g/ml.
2, platelet cracking content and Wnt activator compound
Wnt signal paths play a significant role in terms of the self-renewing for maintaining stem cell, and β-catenin, which can be used as, to be turned
Record the coactivator of the factor, the versatilities related genes such as activation c-myc, Oct4, Sox2, Nanog.When Wnt activator activates Wnt
When signal path, β-catenin can be caused to assemble to maintain stem cells self-renewal ability in nucleus.In addition, Wnt believes
Number access also plays an important role to the proliferation for promoting stem cell.
Wnt activator can maintain the dryness of cell and promote the proliferation of cell, in the culture comprising platelet cracking content
The dryness that Wnt activator is added in base when being largely proliferated when urine derived cell can be maintained to carry out original cuiture, to promote
Cell is preferably proliferated.
Preferably, Wnt activator includes at least one of CHIR 99021, BIO, WNT-3a, R-spondin-2.
Wherein, CHIR 99021 is Wnt activator, IC50For 10nM/6.7nM, the CAS No of CHIR 99021:252917-
06-9, structure are as follows：
BIO is Wnt activator, IC50For 5nM, the CAS No of BIO:667463-62-9, structure are as follows：
Preferably, Wnt activator is preferably CHIR 99021, and contents of the CHIR 99021 in the culture medium is preferably
0.0005~5 μM, more preferably 0.001~0.5 μM.
3, platelet cracking content and ROCK inhibitor compound
Rho associated kinases (ROCK) are used as Rho downstream targets, and important work can be played on cell function by extracellular signal
With, including shrink, it moves, proliferation, differentiation and Apoptosis.Wherein, ROCK mediates film bubble, enhances the contraction of actin, and
Activate Caspase signal cascade and Apoptosis.ROCK inhibitor is the small molecule that Rho associated kinases can be inhibited to act on
Inhibitor.Inhibitor of the ROCK inhibitor as Rho associated kinases, differentiation capable of inhibiting cell and apoptosis, improve stem cell
It survives and maintains its self-renewal capacity.It can be improved when adding ROCK inhibitor in the culture medium comprising platelet cracking content
The adherent ability of cell, the apoptosis for inhibiting urine derived cell to be occurred in latent growth period cell, to be conducive to further carry
The proliferation efficiency of high cell.
Preferably, ROCK inhibitor includes Y-27632, Thiazovivin, Fasudil, GSK429286A, Rk1-1447
At least one of.
Wherein, Thiazovivin is novel ROCK inhibitor, IC50It is 0.5 μM, the survival of stem cell can be promoted.
The CAS No of Thiazovivin:1226056-71-8, structure are as follows：
Y-27632 is a kind of emulative ROCK-I and ROCK-II inhibitor of ATP, acts on ROCK-I and ROCK-II,
Ki is respectively 220nM and 300nM.The CAS No of Y-27632:129830-38-2, structure are as follows：
Fasudil(HA-1077；AT-877) hydrochloride is ROCK-II, PKA, PKG, PKC and MLCK inhibitor, Ki difference
It is 0.33 μM, 1.6 μM, 1.6 μM, 3.3 μM and 36 μM.The CAS No of Fasudil:105628-07-7, structure are as follows：
GSK429286A is selective ROCK1 and ROCK2 inhibitor, IC50Value is 14nM and 63nM.GSK429286A's
CAS No:864082-47-3, structure are as follows：
Rk1-1447 is a kind of effective ROCK1 and ROCK2 inhibitor, IC50Value is 14.5nM and 6.2nM.Rk1-1447
CAS No:1342278-01-6, structure are as follows：
Attempt to be added in inventor's research and development ROCK inhibitor such as Thiazovivin, Y-27632, Fasudil,
GSK429286A, Rk1-1447 etc. and by many experiments, wherein Thiazovivin with platelet cracking content compounding effect most
Good, MTT end values are shown when Thiazovivin is added, and Day7 Proliferation datas are 0.89, and other ROCK inhibitors are compared with this
A value is small, and only 0.85 or smaller.Inventor finally determines and compounds Thiazovivin and platelet cracking content, as urine
Ingredient of the derived cell without heterologous culture medium.
Preferably, ROCK inhibitor is preferably Thiazovivin, and contents of the Thiazovivin in the culture medium is excellent
It is selected as 0.01~30 μM, more preferably 0.1~10 μM.
Preferably, the culture medium also contains epithelical cell growth factor, insulin, hydrocortisone, transferrins, kidney
At least one of upper parathyrine, triiodo thryonine.
Epithelical cell growth factor is a kind of important growth factor, is adjusting cell growth, is surviving, and migration, apoptosis increases
Grow etc. plays a significant role.
Insulin is by the beta Cell of islet in pancreas by endogenous or exogenous material such as glucose, lactose, ribose, essence
The stimulation of propylhomoserin, glucagon etc. and a kind of proteohormone secreted.On the one hand, insulin is combined mediation with IGF-IR
IGF-IR autophosphorylations can activate PI3K, and then PIP3 expression is caused to improve, and finally activate AKT, increase to influence cell
It grows, breaks up, apoptosis and glucose inside cells operating.On the other hand, insulin function is suppressed or will influence when signal deficiency dry
The self-renewal capacity of cell, leads to cell differentiation, is played an important role in terms of the self-renewal capacity for maintaining cell.
Hydrocortisone is the steroid hormone that adrenal cortex generates, and has anti-inflammatory and immunosuppressive action.
Iron ion can promote the fast breeding of cell, the presence of iron ion that can also influence DNA and synthesize, Gene regulation etc., this
Outside, the redox reaction of iron ion can promote the formation of highly reactive form of oxygen, and highly reactive form of oxygen can cause oxidative stress, lipid peroxy
Change, DNA damage simultaneously eventually leads to cell death.Therefore, the shortage of intracellular iron ion or it is excessive cell can be caused it is great
It influences, and transferrins, it is a kind of glycoprotein, the transport of iron ion is mainly responsible in cell culture, followed by combines interior
Source property iron ion, to maintaining the balance of iron to play an important role.
Adrenaline is a kind of hormone and neurotransmitter, and adrenocepter activation can stimulate DNA to synthesize and improve cell
Survival.
Trilute is a kind of thyroid hormone, trilute and 1 knot of Thyroid hormone receptor β
Activation MAPK (ERK1/2) signal path is closed, to promote cell Proliferation.
Preferably, the culture medium contains following components：Platelet cracking content, vitamin C, Wnt activator, ROCK inhibit
Agent, epithelical cell growth factor, insulin, hydrocortisone, transferrins, adrenaline, triiodo thryonine, and basis
Culture medium.
Preferably, each component content is in the culture medium：1~100ng/ml of epithelical cell growth factor, insulin 1~
75 μ g/ml, 1~360ng/ml of hydrocortisone, 0.5~75 μ g/ml of transferrins, 0.1~5 μ g/ml of adrenaline, triiodo first
0.1~200pg/ml of gland original ammonia acid, 10~200 μ g/ml of L-AA, 99,021 0.001~0.5 μ of Wnt activator CHIR
M, 0.1~10 μM of ROCK inhibitor Thiazovivin, platelet cracking content 0.5%v/v~20%v/v, basal medium are mended
Enough to 1L.
Preferably, basal medium includes DMEM, DMEM/F12, α MEM, RPMI 1640, CMRL-1066, Ham's
The above-mentioned basal medium of at least one of F12, IMDM, 199, MCDB or a variety of is mixed with arbitrary proportion.The culture medium does not have
There is concentration requirement, is the uniform concentration of commercial product.
Preferably, basal medium is mixed according to arbitrary proportion by DMEM/F12 or DMEM or DMEM/F12 and DMEM.
A kind of urine derived cell, the cell are obtained using any of the above-described the method culture.
The beneficial effects of the invention are as follows：
Applicant is final to confirm that people's source platelet cracking content is urinated as no heterologous by the way that screening is tested in large quantities for many years
The core additive of culture medium when liquid derived cell culture, existing urine source is thin when can overcome in the past without heterologous culture
Born of the same parents' growth rate is extremely slow and the problems such as cell death gradually occurs.Add people source platelet cracking content without heterologous culture medium
Urine derived cell can preferably be cultivated, studied or clinical application needed for cell concentration.
Platelet cracking content is used for the culture of urine derived cell by the present invention, can be greatly promoted urine derived cell and be lured
It leads to obtain multipotential stem cell and promotes the clinical application of multipotential stem cell.Platelet cracking content of the present invention is in urine derived cell
Application in no heterologous culture and culture medium has novelty, is of great significance to regenerative medicine.
Platelet cracking content is added in urine derived cell is without heterologous culture medium in the present invention, it belong to people source at
Point, avoid dissimilar substances from existing, selected human blood platelets lysate can obtain from healthy blood donor and by immune and disease
Substance detects.Such as fetal calf serum, human blood platelets lysate includes the various necessary factors that can promote cell growth, it contains
TGF-β 1, TGF-β 2, FGF, IGF-1, PDGF-AA, PDGF-AB, PDGF-BB, EGF, VEGF, platelet factor-4 (PF-4),
Attachment element, protease inhibitors, mitogen and coagulation factor can be greatly promoted cell Proliferation.
The present invention is added after platelet cracking content and can be directly separated by many experiments verification in the medium to be urinated
Cell that liquid is included simultaneously is enlarged culture, and experimental result also shows culture medium of the present invention and greatly improves urine source
The growth rate of cell and extend life cycle.
The medium culture that platelet cracking content is added to using the present invention collects to obtain primary urine derived cell, described
Cell is elongated in rice-shaped shuttle, urine derived cell initial incubation to can be observed within the 3rd to 5 day cell adherent, finally may be used
It observes 3 to 5 place's cell clones, passage processing can be carried out to cell at the 15th day, effect is better than the training of 20% fetal calf serum
Support base to the culture effect of urine derived cell (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.)。
The present invention carries out compounding using vitamin C and platelet cracking content and is trained without heterologous for urine derived cell
It supports.Vitamin C can resist the aging for leading to cell due to the increase of frequency dividing cell, it is split comprising blood platelet
Solving addition vitamin C in the culture medium of object can prevent a small amount of urine derived cell from obtaining plurality by a large amount of cell divisions
Irreversible aging occurs after amount cell, vitamin C is cooperateed with platelet cracking content promotes urine derived cell effectively to be held
The cell culture of continuous ground.
Wnt activator can maintain the dryness of cell and promote the proliferation of cell, in the culture comprising platelet cracking content
The dryness that Wnt activator is added in base when being largely proliferated when urine derived cell can be maintained to carry out original cuiture, to promote
Cell is preferably proliferated.
The adherent ability of cell can be improved when adding ROCK inhibitor in the culture medium comprising platelet cracking content, is inhibited
The apoptosis that urine derived cell is occurred in latent growth period cell, to be conducive to further increase the proliferation efficiency of cell.
Description of the drawings
Fig. 1 MTT results；
Fig. 2 MTT results；
Fig. 3 MTT results；
Fig. 4 MTT results；
Fig. 5 MTT results；
Fig. 6 MTT results；
Fig. 7 MTT results；
Fig. 8 MTT results；
Fig. 9 MTT results；
Figure 10 urine derived cells collect culture aspect graph；
Figure 11 urine derived cell culture agings detection figure.
Specific implementation mode
Platelet cracking content in the embodiment of the present invention, it belongs to people source ingredient, dissimilar substances is avoided to exist, selected
Human blood platelets lysate obtained from healthy blood donor and by immune and pathogen detection.
With reference to embodiment, the present invention is described further, but not limited to this.
Embodiment 1
According to table 1 carry out different group culture mediums match liquid, first be added basal medium other than each component, then again plus
Enter basal medium and carry out polishing, wherein basal medium includes DMEM, DMEM/F12, α MEM, RPMI 1640, CMRL-
1066、Ham's F12、IMDM、199、MCDB。
Table 1 is without heterologous urine derived cell culture medium prescription
It digests primary urine derived cell and is counted, per hole (96 orifice plate) 1500 cells of bed board, Day0 uses urine
Liquid primitive cell culture base (REGM culture mediums) is cultivated, Day1, Day3, and Day5 is changed to corresponding group culture medium and is trained
It supports, per 150 μ l of hole culture medium, MTT detections is carried out in Day1, Day4, Day7.
It is examined through MTT, the results are shown in Figure 1 by the MTT of 1-25 groups.
Fig. 1 is the result figure of MTT, and MTT testing principles can make exogenous for the succinate dehydrogenase in living cells mitochondria
MTT is reduced to the bluish violet Jie Jing Jia Za of water-insoluble and is deposited in cell, and dead cell is without this function.DMSO can dissolve carefully
First Za in born of the same parents measures its absorbance value with microplate reader, and within the scope of certain cell number, MTT crystallizes the amount and cell number to be formed
It is directly proportional.According to the absorbance value (OD values) measured, to judge living cells quantity.
From fig. 1, it can be seen that being added to platelet cracking content and each group culture medium without heterologous element can carry out urine
Source cell carries out Multiplying culture, and in Day7, the MTT value highests of group 1, i.e. basal medium are DMEM：DMEM/F12=1:1
More other base culture base effects are good.
Embodiment 2
The present embodiment is to carry out culture medium on the basis of 1 group 1 of embodiment according to table 2 and match liquid, wherein blood platelet is split
The volume ratio that solution object accounts for culture medium is added according to table 3, obtains different groups of other culture mediums.When configuration, basis is first added
Then each component other than culture medium adds basal medium and carries out polishing, wherein basal medium DMEM/F12:
DMEM=1:1.
The 2 platelet cracking content nutrient media components of ratio containing different volumes of table
3 platelet cracking content volume ratio of table
It digests primary urine derived cell and is counted, per hole (96 orifice plate) 1500 cells of bed board, Day0 uses urine
Liquid primitive cell culture base (REGM culture mediums) is cultivated, Day1, Day3, and Day5 is changed to corresponding group culture medium and is trained
It supports, is 150 μ l per hole culture medium, MTT detections are carried out in Day1, Day4, Day7.
It is examined through MTT, the MTT values of 1-18 groups are as shown in Figure 2.
As can be seen from Figure 2, different volumes ratio platelet cracking content can carry out Multiplying culture to primary urine derived cell,
In Day7, group 12, i.e. volume ratio containing platelet cracking content be 10% culture medium MTT value highests.Secondly, it is group
The culture medium MTT values that 10-18, i.e. volume ratio containing platelet cracking content are 8%~20% are taken second place.
Embodiment 3
According to table 4 carry out different group culture mediums match liquid, first be added basal medium other than each component, then again plus
Enter basal medium and carry out polishing, wherein basal medium DMEM/F12:DMEM=1:1.
Platelet cracking content and multivitamin C are added in the culture medium prescription of table 4, vitamin C includes L- Vitamin Cs
Acid, L-Ascorbic Acid L-O-Phosphate.
Table 4 is without heterologous urine derived cell culture medium prescription
It digests primary urine derived cell and is counted, per hole (96 orifice plate) 1500 cells of bed board, Day0 uses urine
Liquid primitive cell culture base (REGM culture mediums) is cultivated, Day1, Day3, and Day5 is changed to corresponding group culture medium and is trained
It supports, per 150 μ l of hole culture medium；MTT detections are carried out in Day1, Day4, Day7.
It is examined through MTT, the results are shown in Figure 3 by the MTT of 1-7 groups.As can be seen from Figure 3, in Day7, be added variety classes and
The vitamin C of various concentration can promote cell Proliferation, wherein the MTT value highests of group 3 in various degree.
Embodiment 4
According to table 5 carry out different group culture mediums match liquid, first be added basal medium other than each component, then again plus
Enter basal medium and carry out polishing, wherein basal medium DMEM/F12:DMEM=1:1.
Table 5 is without heterologous urine derived cell culture medium prescription
It digests primary urine derived cell and is counted, per hole (96 orifice plate) 1500 cells of bed board, Day0 uses urine
Liquid primitive cell culture base (REGM culture mediums) is cultivated, Day1, Day3, and Day5 is changed to corresponding group culture medium and is trained
It supports, per 150 μ l of hole culture medium, MTT detections is carried out in Day1, Day4, Day7.
It is examined through MTT, the results are shown in Figure 4 by the MTT of 1-9 groups, and various concentration L-AA can be to primary urine
Derived cell carries out Multiplying culture, in Day7, group 4, i.e. and the culture medium MTT values of a concentration of 35 μ g/ml containing L-AA
Highest.Secondly, it is group 3-7, i.e. the culture medium MTT values of a concentration of 10~200 μ g/ml containing L-AA are relatively high.It says
The preferred addition content of bright L-AA is 10~200 μ g/ml.
Embodiment 5
According to table 6 carry out different group culture mediums match liquid, first be added basal medium other than each component, then again plus
Enter basal medium and carry out polishing, wherein basal medium DMEM/F12:DMEM=1:1.
Platelet cracking content and a variety of Wnt activator are added in the culture medium prescription of table 6, Wnt activator includes
CHIR99021、BIO、WNT-3a、R-spondin-2。
Table 6 is without heterologous urine derived cell culture medium prescription
It digests primary urine derived cell and is counted, per hole (96 orifice plate) 1500 cells of bed board, Day0 uses urine
Liquid primitive cell culture base (REGM culture mediums) is cultivated, Day1, Day3, and Day5 is changed to corresponding group culture medium and is trained
It supports, per 150 μ l of hole culture medium；MTT detections are carried out in Day1, Day4, Day7.
It is examined through MTT, the results are shown in Figure 5 by the MTT of 1-14 groups.As can be seen from Figure 5, in Day7, variety classes are added
And the Wnt activator of various concentration can promote cell Proliferation, wherein the MTT value highests of group 3 in various degree.
Embodiment 6
According to table 7 carry out different group culture mediums match liquid, first be added basal medium other than each component, then again plus
Enter basal medium and carry out polishing, wherein basal medium DMEM/F12:DMEM=1:1.
Table 7 is without heterologous urine derived cell culture medium prescription
It digests primary urine derived cell and is counted, per hole (96 orifice plate) 1500 cells of bed board, Day0 uses urine
Liquid primitive cell culture base (REGM culture mediums) is cultivated, Day1, Day3, and Day5 is changed to corresponding group culture medium and is trained
It supports, per 150 μ l of hole culture medium, MTT detections is carried out in Day1, Day4, Day7.
It is examined through MTT, the results are shown in Figure 6 by the MTT of 1-9 groups.As can be seen from Figure 6, the CHIR 99021 of various concentration is equal
It can promote the proliferation of urine derived cell, wherein best group is the 5th group, i.e. when 99021 a concentration of 0.025 μM of CHIR
Effect is best, is group 3-8 secondly, i.e., the culture medium MTT values containing 99021 a concentration of 0.001~0.5 μM of CHIR are relatively
It is high.Illustrate that the preferred addition content of CHIR 99021 is 0.001~0.5 μM.
Embodiment 7
Different group culture mediums are carried out according to table 8 and match liquid, and each component other than basal medium is first added, then adds
Basal medium carries out polishing, wherein basal medium DMEM/F12:DMEM=1:1.
Platelet cracking content and a variety of ROCK inhibitors are added in the culture medium prescription of table 8.ROCK inhibitor includes Y-
27632、Thiazovivin、Fasudil、GSK429286A、Rk1-1447。
Table 8 is without heterologous urine derived cell culture medium prescription
It digests primary urine derived cell and is counted, per hole (96 orifice plate) 1500 cells of bed board, Day0 uses urine
Liquid primitive cell culture base (REGM culture mediums) is cultivated, Day1, Day3, and Day5 is changed to corresponding group culture medium and is trained
It supports, per 150 μ l of hole culture medium；MTT detections are carried out in Day1, Day4, Day7.
It is examined through MTT, the results are shown in Figure 7 by the MTT of 1-17 groups.As can be seen from Figure 7, in Day7, variety classes are added
And the ROCK inhibitor of various concentration can promote cell Proliferation, wherein the MTT value highests of group 6 in various degree.
Embodiment 8
Different group culture mediums are carried out according to table 9 and table 10 and match liquid, each component other than basal medium are first added, then again
Basal medium is added and carries out polishing, wherein basal medium DMEM/F12:DMEM=1:1.
Table 9 is without heterologous urine derived cell culture medium fractions
10 thiazovivin various concentrations of table
It digests primary urine derived cell and is counted, per hole (96 orifice plate) 1500 cells of bed board, Day0 uses urine
Liquid primitive cell culture base (REGM culture mediums) is cultivated, Day1, Day3, and Day5 is changed to corresponding group culture medium and is trained
It supports, per 150 μ l of hole culture medium；MTT detections are carried out in Day1, Day4, Day7.
It is examined through MTT, the results are shown in Figure 8 by the MTT of 1-15 groups.As it can be observed in the picture that each group culture medium can carry out urine
Source cell carries out Multiplying culture, and in Day7, the thiazovivin that various concentration is added can be in the increasing for promoting cell in various degree
It grows, wherein the MTT value highests of group 7.
Embodiment 9
Different group culture mediums are carried out according to table 11 and match liquid, and each component other than basal medium is first added, then adds again
Enter basal medium and carry out polishing, wherein basal medium DMEM/F12:DMEM=1:1.
Table 11 is without heterologous urine derived cell culture medium prescription
It digests primary urine derived cell and is counted, per hole (96 orifice plate) 1500 cells of bed board, Day0 uses urine
Liquid primitive cell culture base REGM culture mediums are cultivated, and Day1, Day3, Day5 are changed to corresponding group culture medium and are trained
It supports, per 150 μ l of hole culture medium；Day1, Day4, Day7 carry out MTT detections.
It is examined through MTT, the results are shown in Figure 9 by the MTT of 1-8 groups.As can be seen from Figure 9, each group culture medium can carry out urine
Source cell carries out Multiplying culture, and in Day7, the other MTT of group of vitamin C, CHIR 99021, Thiazovivin are added jointly
It is worth highest.It is compounded using platelet cracking content and vitamin C, platelet cracking content and Wnt activator is compounded and blood
When platelet lysate and ROCK inhibitor are compounded, these three compounding effects are suitable, when platelet cracking content and vitamin C,
When this three of Wnt activator, ROCK inhibitor is carried out at the same time compounding, effect is more than its effect compounded two-by-two.
Embodiment 10
The present embodiment is to carry out primary urine derived cell culture medium using group 8 in embodiment 9 to match liquid.Collect 10ml-
The urine of 2L, 1010g centrifuge 5min, abandon supernatant, be resuspended and carry out centrifuging again abandoning to precipitation with containing dual anti-PBS
Clearly, it then is precipitated to tissue culture plate (24 orifice plate) using urine primitive cell culture base weight is outstanding, and antibiotic is added
primoycin(1:500) 37 DEG C, are then placed into, 5%CO2It is cultivated in cell incubator, during which other is not carried out to it
Operation, carried out changing liquid after five days, waited for that cell clone is grown to a certain size progress had digestive transfer culture.
It is as shown in Figure 10 to cultivate the primary urine derived cell being collected into.It can be seen that cell is elongated in rice-shaped shuttle,
Urine derived cell culture to can be observed within the 3rd to 5 day cell adherent, 3 to 5 place's cell clones finally can be observed,
Passage processing can be carried out within 15 days to cell, effect imitates the culture of urine derived cell better than the culture medium of 20% fetal calf serum
Fruit (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.).Therefore, it can be collected simultaneously with the obtained no heterologous urine derived cell culture medium of liquid according to group 8 in embodiment 9
Culture obtains primary urine derived cell.
Embodiment 11
The present embodiment is to carry out primary urine derived cell culture medium according to each group in embodiment 9 to match liquid.It digests primary
Urine derived cell is simultaneously counted, and per hole (24 orifice plate) 2000 cells of bed board, Day0 uses urine primitive cell culture base
(REGM culture mediums) is cultivated, Day1, Day3, and Day5 is changed to corresponding group culture medium and is cultivated, the addition culture per hole
Base 500 μ l, Day7 carry out aging detection.
When carrying out aging detection, PBS is first added and is washed, is washed with PBS after fixed, is then added again
The sweet enzyme dyeing liquid of beta galactose of Fresh carries out 37 DEG C of overnight incubations, second day microscopically observation colored state.
It is examined through beta galactosidase aging, the coloration result of the day7 of 1-8 groups is as shown in figure 11.As can be seen from Figure 11,
Each group culture medium can carry out urine derived cell Multiplying culture, and cell is more young, wherein coloration result shows the
8 groups of cell is most young.The above experimental data shows：The present invention culture medium 1~100ng/ml of epithelical cell growth factor,
1~75 μ of insulin/ml, 1~360ng/ml of hydrocortisone, 0.5~75 μ of transferrins/ml, 0.1~5 μ g/ of adrenaline
Ml, 0.1~200pg/ml of triiodo thryonine, 10~200 μ g/ml of vitamin C, Wnt activator CHIR 99,021 0.001
~0.5 μM, 0.1~10 μM of ROCK inhibitor Thiazovivin, the concentration model of platelet cracking content 0.5%v/v~20%v/v
In enclosing, it can promote the growth rate of urine derived cell and unicellular survival rate.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications,
Equivalent substitute mode is should be, is included within the scope of the present invention.
Claims (15)
1. a kind of cultural method of urine derived cell, it is characterised in that：Using the culture medium comprising platelet cracking content to urine
Liquid derived cell is cultivated.
2. cultural method according to claim 1, it is characterised in that：The platelet cracking content includes that people source blood platelet is split
Solve the platelet cracking content of object and/or/other source of species.
3. cultural method according to claim 2, it is characterised in that：People source platelet cracking content includes the blood of people
The platelet cracking content that the platelet lysates in source obtain, platelet lysates that multiple types stem cell differentiates and the blood that comes
Platelet lysate, other types cell reprograms transdifferentiation and the platelet lysates come and the platelet cracking content come, or utilizes
Platelet lysates acquired in biological means and at least one of the platelet cracking content come.
4. cultural method according to claim 3, it is characterised in that：The content of platelet cracking content is excellent in the culture medium
It is selected as 0.5%~20%, by volume percentage.
5. cultural method according to claim 4, it is characterised in that：The culture medium also contains vitamin C, Wnt is activated
At least one of agent, ROCK inhibitor.
6. cultural method according to claim 5, it is characterised in that：Vitamin C includes L-AA, vitamin C phosphorus
At least one of acid esters magnesium or its like derivatives.
7. cultural method according to claim 6, it is characterised in that：The content of L-AA is preferred in the culture medium
For 1~700 μ g/ml.
8. cultural method according to claim 5, it is characterised in that：Wnt activator includes CHIR 99021, BIO, WNT-
At least one of 3a, R-spondin-2.
9. cultural method according to claim 8, it is characterised in that：Wnt activator is preferably CHIR 99021, CHIR
99021 content in the culture medium is preferably 0.0005~5 μM.
10. cultural method according to claim 5, it is characterised in that：ROCK inhibitor include Y-27632,
At least one of Thiazovivin, Fasudil, GSK429286A, Rk1-1447.
11. cultural method according to claim 10, it is characterised in that：ROCK inhibitor is preferably Thiazovivin,
Contents of the Thiazovivin in the culture medium is preferably 0.01~30 μM.
12. according to claim 1-11 any one of them cultural methods, it is characterised in that：It is thin that the culture medium also contains epidermis
At least one of the intracellular growth factor, insulin, hydrocortisone, transferrins, adrenaline, triiodo thryonine.
13. according to claim 12 any one of them cultural method, it is characterised in that：The culture medium contains following components：
Platelet cracking content, vitamin C, Wnt activator, ROCK inhibitor, epithelical cell growth factor, insulin, hydrocortisone,
Transferrins, adrenaline, triiodo thryonine and basal medium.
14. according to claim 13 any one of them cultural method, it is characterised in that：Each component content in the culture medium
For：1~100ng/ml of epithelical cell growth factor, 1~75 μ g/ml of insulin, 1~360ng/ml of hydrocortisone, turn iron egg
White 0.5~75 μ g/ml, 0.1~5 μ g/ml of adrenaline, 0.1~200pg/ml of triiodo thryonine, L-AA 10~
200 μ g/ml, 99,021 0.001~0.5 μM of Wnt activator CHIR, 0.1~10 μM of ROCK inhibitor Thiazovivin, blood
Platelet lysate 0.5%v/v~20%v/v, basal medium complement to 1L.
15. a kind of urine derived cell, it is characterised in that：The cell is trained using any one of claim 1-14 the method
It supports and obtains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2018/101079 WO2019109666A1 (en) | 2017-12-05 | 2018-08-17 | Method for culturing urine-derived cells |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711267508 | 2017-12-05 | ||
| CN2017112675088 | 2017-12-05 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108570446A true CN108570446A (en) | 2018-09-25 |
Family
ID=63576649
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810067658.2A Pending CN108570446A (en) | 2017-12-05 | 2018-01-24 | A kind of cultural method of urine derived cell |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN108570446A (en) |
| WO (1) | WO2019109666A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305835A (en) * | 2019-06-26 | 2019-10-08 | 中山大学附属第一医院 | Kidney transplant donor specific urinates the preparation method and its application of source cell and its DNA |
| CN112458037A (en) * | 2020-11-25 | 2021-03-09 | 四川大学华西医院 | Urine cell culture method |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20220065805A (en) * | 2019-09-20 | 2022-05-20 | 에모리 유니버시티 | Endothelial and smooth muscle, such as tissue produced from urinary cells, and uses related thereto |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701671A (en) * | 2016-11-14 | 2017-05-24 | 天津市康婷生物工程有限公司 | Culture system beneficial for mesenchymal stem cells to be applied in bone regeneration |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2069479A1 (en) * | 2006-09-18 | 2009-06-17 | Medizinische Universität Graz | Plasma-free platelet lysate for use as a supplement in cell cultures and for the preparation of cell therapeutics |
| CN105505854B (en) * | 2016-01-14 | 2019-07-12 | 上海市第六人民医院 | Acquisition methods and application from the excretion body of human urine cell |
-
2018
- 2018-01-24 CN CN201810067658.2A patent/CN108570446A/en active Pending
- 2018-08-17 WO PCT/CN2018/101079 patent/WO2019109666A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106701671A (en) * | 2016-11-14 | 2017-05-24 | 天津市康婷生物工程有限公司 | Culture system beneficial for mesenchymal stem cells to be applied in bone regeneration |
Non-Patent Citations (3)
| Title |
|---|
| 华杰等: ""血小板裂解液替代胎牛血清培养间充质干细胞"", 《中华实验外科杂志》 * |
| 张文成等: ""间充质干细胞无异源/无血清培养基研发的现状和前景"", 《中国医药生物技术》 * |
| 牛鑫等: ""人类尿液来源干细胞的体外培养和生物学特性分析"", 《实验与检验医学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110305835A (en) * | 2019-06-26 | 2019-10-08 | 中山大学附属第一医院 | Kidney transplant donor specific urinates the preparation method and its application of source cell and its DNA |
| CN112458037A (en) * | 2020-11-25 | 2021-03-09 | 四川大学华西医院 | Urine cell culture method |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2019109666A1 (en) | 2019-06-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Xu et al. | A zebrafish embryo culture system defines factors that promote vertebrate myogenesis across species | |
| EP2956538B1 (en) | Bioengineered liver constructs and methods relating thereto | |
| CN105296418B (en) | A kind of method and its application of long-term in vitro culture and amplification liver cell | |
| CN108570443A (en) | A kind of culture medium for cultivating urine derived cell | |
| EP3031908A1 (en) | Method for producing dopaminergic neurons | |
| CN103834613B (en) | The method for preparing multipotency angiocarpy precursor and maintaining its cardiovascular differentiation capability | |
| Lee et al. | Effects of redox modulation on cell proliferation, viability, and migration in cultured rat and human tendon progenitor cells | |
| Becker et al. | Divergent fate and origin of neurosphere-like bodies from different layers of the gut | |
| Li et al. | Construction of bioengineered hepatic tissue derived from human umbilical cord mesenchymal stem cells via aggregation culture in porcine decellularized liver scaffolds | |
| CN108570446A (en) | A kind of cultural method of urine derived cell | |
| Zhou et al. | Characterization and standardization of cultured cardiac fibroblasts for ex vivo models of heart fibrosis and heart ischemia | |
| Tang‐Schomer et al. | In vitro 3D regeneration‐like growth of human patient brain tissue | |
| JP2019089846A (en) | Modulation of angiogenesis | |
| Gheller et al. | Isolation, culture, characterization, and differentiation of human muscle progenitor cells from the skeletal muscle biopsy procedure | |
| Quintero Sierra et al. | Highly pluripotent adipose-derived stem cell–enriched nanofat: a novel translational system in stem cell therapy | |
| CN102282250A (en) | Method of differentiating mammalian progenitor cells into insulin producing pancreatic islet cells | |
| Li et al. | In vitro and in vivo study on angiogenesis of porcine induced pluripotent stem cell-derived endothelial cells | |
| CN110511901A (en) | Bio-artificial proximal tubule system and application method | |
| US20100158873A1 (en) | Method for extracting and selecting cells | |
| WO2020097440A1 (en) | Methods of predicting functional recovery of tissue using circulating exosomes derived from transplanted cells | |
| Bertolo et al. | Comparative characterization of canine and human mesenchymal stem cells derived from bone marrow | |
| CN104755610B (en) | Fat tissue cell | |
| Ho et al. | A serum free approach towards the conservation of chondrogenic phenotype during in vitro cell expansion | |
| Boyce et al. | Assessment of replication rates of human keratinocytes in engineered skin substitutes grafted to athymic mice | |
| JP2022545376A (en) | Method for in vitro production of hyaline cartilage tissue |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180925 |
|
| RJ01 | Rejection of invention patent application after publication |