CN107904207A - A kind of method for obtaining mature hepatocytes using three dimensional culture system induction people inductive pluripotent stem cells - Google Patents

A kind of method for obtaining mature hepatocytes using three dimensional culture system induction people inductive pluripotent stem cells Download PDF

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CN107904207A
CN107904207A CN201711126491.4A CN201711126491A CN107904207A CN 107904207 A CN107904207 A CN 107904207A CN 201711126491 A CN201711126491 A CN 201711126491A CN 107904207 A CN107904207 A CN 107904207A
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stem cells
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于云飞
彭特
陈勇
蔡亚雄
乔志平
刘樱
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Guangdong Xtem Biotechnology Co Ltd
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Abstract

The present invention provides a kind of method that people's inductive pluripotent stem cells efficiently, are in heterogeneity induced to differentiate into liver cell using three dimensional culture system, it is to utilize three-dimensional extracellular matrix culture stent made of polylactide glycolic acid copolymer (PLGA), and/or a variety of inducible factors of joint include the liver cell that definitive endoderm inducible factor, hepatocyte growth factor etc. induce people's inductive pluripotent stem cells to be divided into maturation.The present invention establishes a kind of method of new acquisition people's mature hepatocytes, people's inductive pluripotent stem cells are efficiently induced to be divided into ripe liver cell using 3 D stereo culture systems and/or a variety of inducible factors of joint first, liver cell is induced to differentiate into for people's inductive pluripotent stem cells technical support is provided, and improve cell material for the research in the fields such as the mechanistic research of hepatopathy, drug screening.

Description

One kind obtains ripe liver using three dimensional culture system induction people inductive pluripotent stem cells The method of cell
Technical field
The present invention relates to biological technical field, and in particular to a kind of side that mature hepatocytes are obtained using three dimensional culture system Method.
Background technology
Liver failure serious threat human health, and lethality is high.Liver Transplantation for Treatment is current optimal treatment liver The means of exhaustion, but due to the shortage of donor, popularity can not be carried out.Artificial Liver Support System can temporarily substitute liver work( Can, help patient to spend hepatic failure critical days and wait liver cell regeneration, or maintain patient to survive to obtaining suitable donor Carry out liver transfer operation.Bioartificial liver is to be presently considered to optimal artificial liver, but its development is limited to the limit of hepatocyte origin System.Human pluripotent stem cells, including embryonic stem cell and inductive pluripotent stem cells, can be long under appropriate condition of culture in vitro Phase largely expands, and includes the potential of candidate stem cell with all cell types that needed by human body is wanted are divided into, and is a kind of solution Certainly mature hepatocytes carry out the method for source problem.Before the development of IPS cell researches and its clinical practice in cell replacement therapy Scape provides powerful technical support for bioartificial liver.
2009, Song etc. first by successively addition Activin A, fibroblast given birth in the document delivered by report The long factor 4 (fibroblast growth factor 4, FGF4) and bone morphogenetic protein2 (bone morphogenetic Protein 2, BMP2) etc. the factor induced by four-stage from iPS cells except the cell of similar liver cell sample.Takata The Activin A culture 3d of high dose are firstly added by " two-step method " Deng report in 2011, induce iPS cells embryo into sizing Confluent monolayer cells convert, and by adding HGF after endoderm cell is obtained, continue to cultivate 5d, liver can be also obtained by two-step method Cell-like cell.2012, the report such as Chen induced iPS cells to obtain mature hepatocytes using " three-step approach ", and the first step exists Activin A, Wnt3 α and HGF are added in iPS cell culture mediums；Second step, using liver sizing culture medium, further culture is thin Born of the same parents；3rd step, in IMDM culture mediums, while adds and contains carcinophylin albumen M, dexamethasone and universal culture medium, realize It is in vitro hepatocyte-like cells by the induction of iPS cells, and induced efficiency significantly improves.
Polylactic acid (polylactic acid, PLA) and polyglycolic acid (polyglycolic acid, PGA) are most allusion quotations The synthesized degradable polymer of type, and the simplest linear polyhydroxyalkanoate of structure.They are degradable as first Absorbing material is approved by the fda in the United States for clinic, is studied so far most extensively, using most degradable biomaterials.PLGA Performance mechanical property, degradation speed and fibroblastic biologically can be produced due to the difference of PLA and PGA ratios Difference.And the PLGA of proper ratio can provide the environment that can stick as the stent of cell growth for the growth of cell. Inducible factor cell growth, differentiation, the stimulation of metabolism can be increased again by combining inducible factor on PLGA stents.
The content of the invention
It is an object of the present invention to provide it is a kind of using three dimensional culture system obtain mature hepatocytes method, by with Lower technical solution is realized：With mechanical passage method (glass draws pin segmentation, picking) people's inductive pluripotent stem cells, illustrated according to reagent Book handles the Three-dimensional cell culture materials such as various polylactic acid, polyglycolic acid material, and/or the related liver of joint lures to differentiation (including definitive endoderm inducible factor-Wnt3 α, activin A, liver is to entoderm inducible factor-Bone Morphogenetic Protein 4, alkali for inducement Property fibroblast growth factor, hepatic progenitor cells inducible factor-hepatocyte growth factor, hepatocyte maturation inducible factor-liver are thin The intracellular growth factor), by the induction and culture of 15-30 days, the method for forming mature hepatocytes.
Mature hepatocytes are to induce differentiation to obtain by people's inductive pluripotent stem cells.
People's inductive pluripotent stem cells are to utilize to contain transcription factor pMXs-OCT4, pMXs-SOX2, pMXs- The retrovirus induction human foreskin fibroblasts of tetra- kinds of factors of KLF4 and pMXs-c-MYC, reprogram people's inductivity of acquisition Multipotential stem cell.
The three-dimensional cell cultivation material includes a variety of Three-dimensional cell culture matrix or cell culturing bracket, has such as wrapped up not The polylactic acid of the isogeneous induction factor, the PLGA microballoons of polyglycolic acid different proportion or PLGA microsphere supports.
Aided in during mature hepatocytes induce in inducing culture addition inducible factor cancer suppressor protein M, fill in rice Pine and transferrins, these types of inducible factor are not united with PLGA microsphere supports, but by adding in the medium Mode aid in promote hepatocyte maturation.
Albumin in enzyme linked immunological enzyme linked immunosorbent assay (Elisa), PCR methods, immuno-fluorescence assay mature hepatocytes Induction Process (ALB), the expression of the Marker such as cytokeratin (CK-18, CK-19).
It is characteristic of the invention that utilize a kind of biodegradable Three-dimensional cell culture system, and/or a variety of inductions of joint The induced multi-potent stem cells such as the factor, hepatocyte maturation factor are divided into mature hepatocytes, establish and efficiently obtain mature hepatocytes Method.This method provides reliable cell derived to obtain useful clinically liver cell, establishes utilize dimensional culture first The system that efficiently induced multi-potent stem cell is divided into mature hepatocytes such as system and the/a variety of factors, for obtain it is useful clinically into Ripe liver cell provides theoretical foundation and technology platform, and is visited for the mature hepatocytes in multipotential stem cell source in pathogenic mechanism New method and new thinking have been opened up in the application in the fields such as rope, drug screening.
Brief description of the drawings
Attached drawing used is with PLGA microsphere support culture human pluripotent stem cells, joint Wnt 3 α and Activin in the present invention A inducible factors, and the factor induced multi-potent stem cell such as addition in the medium BMP4, HGF and bFGF aided in is divided into maturation Liver cell experimental data is representative, is illustrated.
Fig. 1 is the three-dimensional induction differentiation scheme of mature hepatocytes in multipotential stem cell source.
Fig. 2 is that human pluripotent stem cells occur changing (1-2 generations) to the cellular morphology of passage from clone.
Fig. 3 is the part specificity multipotency Marker expressions of multipotential stem cell.
Fig. 4 is PLGA microballoons and PLGA microsphere support form electron microscopes.
Fig. 5 is human pluripotent stem cells absorption growthform figure on PLGA microballoons.
Fig. 6 is the hepatocyte-like cells growthform figure that can be passed on after PLGA microsphere supports are degraded.
Fig. 7 is mature hepatocytes part functionalities marker identification figure.
Embodiment
Below according to attached drawing, the present invention is further illustrated in conjunction with specific embodiments, to more fully understand the present invention. Material used, reagent etc., are commercially available unless otherwise specified in embodiment.
Embodiment three dimensional culture system induced multi-potent stem cell is divided into mature hepatocytes
The present invention provides utilize the Three-dimensional cell culture materials such as Three-dimensional cell culture material polylactic acid, polyglycolic acid material Handled, and/or the related liver of joint to differentiation induction factor (including definitive endoderm inducible factor-Wnt3 α, activin A, Liver is to entoderm inducible factor-Bone Morphogenetic Protein 4, basic fibroblast growth factor, hepatic progenitor cells inducible factor-liver cell Growth factor, hepatocyte maturation inducible factor-hepatocyte growth factor), by the induction of 15-30 days, obtain mature hepatocytes Method.Concrete scheme is referring to Fig. 1.
1. the culture of multipotential stem cell, amplification and passage
1) the hiPS cells frozen are taken out in recovery, are put in 37 DEG C of warm water and quickly rock, make ice crystal fast melt, add dropwise Enter 10 times of volumesCulture medium, mixing is gently blown and beaten with pipettor, 800rpm/min centrifugation 5min, abandon supernatant, add Enter the fresh mTeSR culture mediums of 4mL, piping and druming mixes, and is added in advance with six orifice plates of treated 30min.One was changed every 2 days Subculture.
2) 1. type Ⅳ collagenase had digestive transfer culture method is passed on：Treat that colony degrees of fusion reaches 90% or so, or occur " jaundice " or During noble cells, first with glass pin can be drawn to remove noble cells, DPBS is cleaned once, with the type Ⅳ collagen enzymic digestion of 1mg/mL 15-20min, colony is blown down, and is added 3-5mL culture mediums, is gently dispelled into microcolony, is uniformly inoculated in Matrigel pretreatments Culture plate or ware on；2. mechanical passage method：The clone that will need to pass under inverted phase contrast microscope again before passage carries out mark Remember, draw pin to clone and (be chiefly used in single larger clone's passage) mechanically decoupled to suitable size with glass under aseptic condition, then Suctioned out, be seeded on the plate of Matrigel pretreatments with pipettor.Using mTeSR culture mediums, replace once within 2 days.
3) the hiPS cell colonies that will be digested are frozen to be transferred in centrifuge tube, are gently blown with pipettor or pasteur pipet Beat to suitable size, 800rpm/min centrifugation 5min, abandon supernatant, add prepared frozen stock solution (70%mTeSR culture mediums in advance + 10% cell culture level DMSO of+20% serum substitute), mix, marked title, date and generation, be placed on program cooling Stayed overnight for -80 DEG C in box, next day is transferred in -200 DEG C of liquid nitrogen containers and preserves.
2. the expression identification of human pluripotent stem cells specificity marker
By hiPS cell inoculations on 24 orifice plates or creep plate, treat that cell fusion degree is 70% or so or clonal growth to " side Edge is obvious " after, to take out from strong fragrant perfume, rinsed 3 times, each 3min with the PBS of pre-temperature, 4% formaldehyde room temperature fixes 15min, PBS washes 3 times, each 3min, 0.5%Triton X-100 permeabilizations 15min, and PBS is washed 3 times, each 3min, 5%BSA room temperatures envelope 30min is closed, blotting paper sops up confining liquid, adds straight mark (OCT3/4) or mark (Nanog, SOX2) fluorescence primary antibody is placed on wet box In, 4 DEG C of overnight incubations, PBS is washed 3 times, each 3min, and dropwise addition fluorescence secondary antibody (directly marks fluorescence after blotting paper blots unnecessary PBS This step is not required in antibody OCT3/4), 37 DEG C are incubated 1h (lucifuge processing), and PBS is washed 3 times, each 3min, redyes DAPI, lucifuge is incubated 1min is educated, PBS is washed 5 times, each 2min, mountant mounting.Fluorescence microscopy Microscopic observation.
3. the preparation of microsphere support
1) prepared by microballoon is dissolved in 4mL CH by 1g PLGA2Cl2In, ultrasonic vibration 15min is until PLGA is completely dissolved.Claim Take 0.4g β-TCP dispersed in a liquid.Weigh 3g PVA and be placed in 300mL deionized waters and heat and be stirred continuously, speed For 300rpm/min, the PLGA solution containing β-TCP is slowly added dropwise after PVA is completely dissolved, be stirred continuously during dropwise addition into And generate microballoon.After stirring 6h, CH is treated2Cl2Evaporate into the greatest extent, take out microballoon, No. 5 microballoons are cleaned with deionized water, then by microballoon into Row frozen dried, removes the moisture in microballoon, obtains dry microballoon.
2) stent is prepared experiment microsphere support and is prepared using sintering process, and microballoon is sieved, and it is 300- to select particle diameter 450 μm of microballoon, microballoon is banged into a diameter of 5mm in bottom, highly for 10mm cylindrical mold in, then by equipped with microballoon Mould heats 1h in 70 DEG C of baking oven, and mould is taken out after heating, makes its cooled to room temperature, removes mould, obtains Stent.
4. establish the system that efficiently induction human pluripotent stem cells are divided into mature hepatocytes using Three-dimensional cell culture system
1) type Ⅳ collagen enzymic digestion multipotential stem cell.With type Ⅳ collagen enzymic digestion multipotential stem cell 15-30min, glue is discarded Protoenzyme, adds 3mL mTeSR culture mediums and terminates digestion, and centrifugation, abandons supernatant, by cell precipitation piping and druming for small cell cluster (as far as possible not It can be separated into unicellular).
2) suitable cell is taken to be resuspended in mTeSR culture mediums, adjustment cell density is 5 × 106A cell/mL, The cell suspending liquid of 2 × working concentration is made.
3) PLGA microsphere supports suspension and pluripotent cell suspension are mixed in equal volume.
4) gently shake up, cell suspending liquid is sufficiently mixed with PLGA microsphere support suspension, stand 37 DEG C, 5%CO2 Cultivate 48h.
5) in the experimental group containing inducible factor, different phase adds corresponding inducible factor.Definitive endoderm (DE) Differential period, inducing culture are to be added in DMEM (Dulbecco's Modified Eagle Medium) basal medium 10% hyclone (FBS), 100ng/mL Activin A, 3 α of 50ng/mL Wnt, cultivate 5-7d, and replacement in every 2 days is once trained Support base.To liver to entoderm (hepatic endoderm) stage, inducing culture is to add in DMEM basal mediums for DE differentiation Enter the bFGF (FGF-2) of 10%FBS, 50ng/mL BMP4,20ng/mL, cultivate 5-7 days, replace a subculture within every 2 days. It is DMEM bases that Hepatic endoderm, which break up to hepatic progenitor cells (immature hepatocytes) stage, inducing culture, Basal culture medium adds 2%FBS, 20ng/mL HGF, cultivates 5-9 days, changes liquid within every two days.Hepatocyte maturation (Hepatocyte Maturation) stage, inducing culture add 2%FBS, 20ng/mL HGF, 20ng/mL for DMEM basal mediums Oncostatin M, 50nM dexamethasone, 1 × insulin Transferrin solution culture 15d-17d, change liquid in every 2 days.
5. mature hepatocytes identified by immunofluorescence
Identification method is identical with the expression identification method of 3 human pluripotent stem cells specificity markers, and fluorescence primary antibody is changed to FOXA2 (forkhead box A2) fluorescence primary antibody, alpha-fetoprotein (alpha-fetoprotein, AFP) fluorescence primary antibody, liver cell Nuclear factor (hepatocyte nuclear factor 4 α, HNF4 α) fluorescence primary antibody and albumin (Albumin) fluorescence primary antibody.
By above example the results show that can be induced human pluripotent stem cells using the three dimensional culture system of the present invention Hepatic progenitor cells and ripe sample liver cell are divided into, and induced efficiency is improved by dimensional culture system, shortens induction duration, institute State the mature hepatocytes that ripe sample liver cell can be identified as having liver function by identification.Above to the specific reality of the present invention Example is applied to be described in detail, but it is intended only as example, and the present invention is not restricted to the specific embodiment of description.For this For field technology personnel, any equivalent modifications carried out to the present invention and replacement are also all among scope of the invention.Therefore, The impartial conversion made without departing from the spirit and scope of the invention and modification, all should be contained within the scope of the invention.

Claims (6)

  1. A kind of 1. method that mature hepatocytes are obtained using three dimensional culture system, it is characterised in that this method passes through following technology Scheme is realized：
    (1) the hiPS cells that recovery freezes, with the segmentation of mechanical passage method, picking people's inductive pluripotent stem cells；
    (2) three dimensional culture system is established, different phase adds corresponding inducible factor, and efficiently induction people's inductivity is more stage by stage Can dry cell differentiation acquisition mature hepatocytes；
    (3) pass through the induction and amplification of 15-30 days, obtain people's mature hepatocytes with subtotal hepatectomy.
  2. 2. according to the method described in claim 1, it is characterized in that, the three dimensional culture system combines a variety of three-dimensional cell trainings Support matrix or cell culturing bracket and induced differentiation factor and growth factor.
  3. 3. method according to claim 1 or 2, it is characterised in that the Three-dimensional cell culture matrix or cell culture Stent selection by various concentrations, ratio PLGA made of Three-dimensional cell culture material.
  4. 4. method according to claim 1 or 2, it is characterised in that the induced differentiation factor and growth factor includes Activin A, Wnt3 α, Bone Morphogenetic Protein 4, basic fibroblast growth factor, hepatocyte growth factor, carcinophylin albumen with And dexamethasone.
  5. 5. according to the method described in claim 1, it is characterized in that, the induction hiPS cells are divided into four according to cell factor Stage：
    (1) the first stage three-dimensional microsphere support associational cells factor is together trained for activin A, Wnt3 α and induced multi-potent stem cell Support, cell is fully climbed on microsphere support, induce as definitive endoderm；
    (2) cell factor that second stage is added in the medium is Bone Morphogenetic Protein 4, basic fibroblast growth factor；
    (3) phase III is added to hepatocyte growth factor in the medium；
    (4) also supplement adds carcinophylin albumen and dexamethasone to fourth stage in addition to HGF.
  6. 6. according to the method described in claim 1, it is characterized in that, the hiPS cells are by containing transcription factor The retrovirus mode of tetra- kinds of transcription factors of OCT4, SOX2, KLF4 and c-MYC induces human foreskin fibroblasts reprogramming to obtain The inductive pluripotent stem cells obtained.
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CN111394297A (en) * 2018-12-17 2020-07-10 中国科学院分子细胞科学卓越创新中心 Method for preparing functional hepatoblasts and liver parenchymal cells from endoderm stem cells in large scale and application of method
CN111621467A (en) * 2020-06-12 2020-09-04 青岛大学 Method for inducing human adipose-derived mesenchymal stem cells to differentiate into liver-like cells
CN115011550A (en) * 2022-07-14 2022-09-06 广东乾晖生物科技有限公司 Induction medium and method for inducing differentiation and maturation of liver cells under suspension culture condition

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