CN106148270B - A kind of construction method of the micro- hepatic tissue unit of three-dimensional for biological artificial liver support system - Google Patents
A kind of construction method of the micro- hepatic tissue unit of three-dimensional for biological artificial liver support system Download PDFInfo
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Abstract
The invention discloses a kind of construction methods of micro- hepatic tissue unit of three-dimensional for biological artificial liver support system.It is characterized in that taking off cytoskeletal elements using sodium alginate-polycation preparation microencapsulation pig liver, and compound liver cell, vascular endothelial cell and fibroblast, by cell self assembly come the three-dimensional micro- hepatic tissue unit of constructing function, and it is applied to Biotype artificial liver.Operation of the present invention is simple, it is at low cost, pass through the interaction in analogue body in hepatic tissue microenvironment between cell and extracellular matrix, cell and soluble factor and cell and cell, so that liver cell, vascular endothelial cell and fibroblast are self-assembled into three-dimensional micro- hepatic tissue, have the function of good synthesis, secretion and removing toxic substances etc., so as to be applied in biological artificial liver support system as micro unit.
Description
Technical field
The present invention relates to biological artificial liver support system field, specifically a kind of three for biological artificial liver support system
Tie up the construction method of micro- hepatic tissue unit.
Background technique
Liver is the most important metabolic organ of human body, and bioartificial liver (Bio-artificial liver, BAL) supports system
System can preferably substitute the synthesis of liver, the functions such as metabolism and secretion, and the substituted or supplemented method as orthotopic liver transplantation is shown
Good application prospect is shown.However, the liver cell under conventional two dimension condition of culture loses the main function such as its synthesis, removing toxic substances quickly
Can, therefore how to obtain high function liver cell is that bioartificial liver applies faced one of key problem.
It is a variety of thin in hepatic tissue microenvironment studies have shown that close regulation of the function of liver cell by hepatic tissue microenvironment
Born of the same parents coexist, and in addition to hepatic parenchymal cells, there are also the nonparenchymal cells such as sinusoid vascular endothelial cell, stellate cells, variety classes
There are important interactions between cell, they, which form three-dimensional hepatic tissue, has activity more higher than independent liver cell and function
Energy.In addition, also containing there are many important extracellular matrix components in hepatic tissue microenvironment, such as collagen, fibronectin, matrix at
Divide other than playing supporting function, important regulating and controlling effect is also played by function of the signal of interest such as integrin to liver cell.
Thus, it can be known that compared with conventional two dimension culture, can simulate between variety classes cell and phase between cell and matrix components
The dimensional culture system of interaction will be hopeful that the micro- hepatic tissue of three-dimensional of similar internal hepatic tissue can be generated, and preferably keep liver
The polarity and normal physiological function of cell, this will promote the development of bioartificial liver system significantly.
However, there is also two critical problems for three-dimensional hepatocyte cultures system existing at present.Firstly, existing system is big
A kind of stroma cell is only used to support liver cell more, it cannot a variety of important cells coexist in hepatic tissue in analogue body well
Feature, it is difficult to form the three-dimensional micro- hepatic tissue of high function.In addition, existing system mainly passes through the ingredients such as addition collagen, hyaluronic acid
The biological function of bracket is improved, however these ingredients fall far short with internal liver cell epimatrix actual constituent, it is difficult to play foot
Enough supports and regulating and controlling effect.
Above-mentioned two problems limit three-dimensional hepatic tissue building and the application of biological artificial liver support system, therefore this hair
Bright propose takes off the micro- hepatic tissue construction method of three-dimensional that cell biomimetic scaffolds and many cells co-culture, the method energy based on hepatic tissue
The characteristics of effectively simulating hepatic tissue microenvironment, can prepare the H.D micro- hepatic tissue of three-dimensional, can be applied to artificial liver system
In system.The present invention, which has the special feature that, is: endothelial cell, fibroblast co-culture with liver cell simultaneously, simulate in hepatic tissue
Various kinds of cell coexists；Hepatic tissue take off cytoskeleton use remain internal hepatic tissue matrix components and a variety of growths because
Son, the single component effect than adding at present are more preferable；Many cells co-culture and being used in combination for de- cytoskeleton is preferably embodied
The main feature of hepatic tissue microenvironment promotes the formation of three-dimensional function micro- hepatic tissue.
Summary of the invention
The purpose of the present invention is to provide a kind of structures of micro- hepatic tissue unit of three-dimensional for biological artificial liver support system
Construction method, it is characterised in that: prepare pig liver first and take off cytoskeleton particle, pig liver obtained is taken off into cytoskeleton later
Grain is uniformly mixed in sodium alginate soln, and liver cell, vascular endothelial cell and fibroblast are then added into mixed liquor,
Calcium chloride solution is instilled later and prepares calcium alginate microsphere, and Solid core alginic acid is made after adding said polycation solution film forming
Sodium-polycation microcapsules adds cell culture fluid in 37 DEG C, contains 5%CO2Static culture system or bioreactor carry out
The building of functional three-dimensional micro- hepatic tissue unit is realized in culture by cell self assembly.The above method of the present invention passes through following tool
Body technique scheme is achieved:
1. pig liver takes off cytoskeleton particle preparation:
Healthy adult Experimental Miniature Pig is selected, is anaesthetized, Men Jing anticoagulant through hepatic arterial infusion heparin solution after opening operation abdomen
Arteries and veins is perfused 37 DEG C through peristaltic pump after being intubated and fixing and contains 0.25% (w/v) trypsase and 0.02% (w/v) ethylenediamine tetra-acetic acid
(EDTA) phosphate buffer to liver is in khaki, and detachment liver peripheral vessels take complete left lateral lobe of liver and right siphonal lobe
Freeze in -80 DEG C >=for 24 hours after, lobe of the liver is cut into 1-4mm thickness with slicer, suitable size tissue, by hepatic tissue piece and de- thin
Cytosol is placed in 4 DEG C of constant-temperature tables in the mixing of 1:15-1:20 (w/v) ratio.De- cell tissue piece uses ball after vacuum freeze drying
Mill instrument is crushed into 10 μm of -100 μm of particles, freezes after gamma-ray irradiation sterilizes spare in -80 DEG C.
2. sodium alginate-polycation, which prepares microencapsulation liver, takes off cytoskeleton particle and cell:
Under aseptic condition, liver is taken off into cytoskeleton particle and is mixed in sodium alginate soln, by liver cell, blood vessel endothelium is thin
Born of the same parents and fibroblast are suspended from proportion in the sodium alginate mixed liquor of the particle of acellular matrix containing liver, are instilled in calcium chloride solution
Calcium alginate microsphere is prepared, is added after said polycation solution film forming through being prepared into solid-state core with excess charge in sodium alginate soln
Heart sodium alginate-polycation microcapsules, addition cell culture fluid are cultivated.
The sodium alginate soln concentration effective range is 2%-4% (w/v)；The molecular weight of sodium alginate is 100-
1000kDa, guluronic acid and mannuronic acid ratio (G/M) range are 0.2-3:1 in sodium alginate molecule；The alginic acid
Sodium is the sodium alginate of arginyl-glycyl-aspartic acid (RGD) modification, Tyrosine-Isoleucine-glycine-serine-
The peptide modified sodium alginate of arginine (YIGSR), Isoleucine-lysine-valine-alanine-valine (IKVAV) polypeptide
It is the mixing sodium alginate of 1:1:1 that sodium alginate, which is modified, with mass ratio.
The polycation includes α-polylysine, poly ornithine, epsilon-polylysine, chitosan, wherein α-polylysine
Molecular weight is 2-80kDa, and Valid concentration is 0.05%-0.2% (w/v)；Poly ornithine molecular weight is 2-80kDa, effectively
Concentration range is 0.05%-0.2% (w/v)；Epsilon-polylysine molecular weight is 5-50kDa, Valid concentration 0.05%-
0.2% (w/v)；Molecular weight of chitosan is 0.5-20kDa, and Valid concentration is 0.05%-0.5% (w/v)；Polycation is molten
The ratio of liquid and the sodium alginate of particle containing acellular matrix suspension is 1:5-1:20 (w/v).
The preparation that the pig liver takes off cytoskeleton particle includes following two process:
Pig liver tissue is sliced preparation process: containing 0.25% (w/v) trypsase and 0.02% (w/v) ethylenediamine with 37 DEG C
The phosphate-buffered perfusion test tube of hepari of tetraacethyl (EDTA) is dissociated left and right -80 DEG C of siphonal lobe warp complete in body liver to khaki
Freeze >=slice is 1-4mm thick tissue piece afterwards for 24 hours；
The de- cell treatment process of liver organization piece: liver organization piece and de-cell liquid are mixed with 1:15-1:20 (w/v) ratio
It closes, de-cell liquid is followed successively by distilled water for 24 hours, 2% (v/v) Triton X-100 and 0.1% (v/v) ammonia spirit 96h, and 0.1%
(w/v) lauryl sodium sulfate (SDS) solution for 24 hours, distilled water 72h, vacuum freeze drying.Cell tissue will be taken off after freeze-drying
Piece, which is placed in ball milling instrument, is ground into the particle that particle size range is 10 μm -100 μm, freezes after gamma-ray irradiation sterilizes in -80 DEG C
It is spare.
It is 0.2%-5% (w/ that the pig liver, which takes off concentration effective range of the cytoskeleton particle in sodium alginate soln,
v)。
The liver cell includes people and mammal (such as pig, rabbit, monkey, rat, mouse) source primary hepatocyte, does carefully
One of born of the same parents' derived hepatocytes, tumorigenic liver cell line, unicellular or liver cell aggregation of immortalized hepatocyte strain；Institute
Stating vascular endothelial cell includes one of people and mammal source Artery, Vein blood endothelial cell strain；It is described at fiber
Cell includes one of the embryo fibroblast of people and mammal source, skin fibroblasts；
The liver cell and vascular endothelial cell number proportional region are 5:1-1:3 (number ratio), liver cell at fiber
Number of cells proportional region is 5:1-1:3 (number ratio), total cell connecing in the sodium alginate suspension of particle containing acellular matrix
Kind density is 1 × 106-1×107A/mL.
The calcium chloride solution concentration is 0.05-0.2mol/L, calcium chloride solution and the alginic acid of particle containing acellular matrix
The ratio (v/v) of sodium suspension is 1:50-1:200, and the calcification time is 15-30min.
The polycation film formation time is 10-20min；The diameter range of the Solid core micro-capsule is 200-2000 μ
m。
The Biotype artificial liver includes that simple Biotype artificial liver and mixed biology artificial liver support system
System, type of reactor includes stirring-type, filling type, hollow fiber form, gas-lifting type, rotary reactor.
The present invention has the advantage that
1. preparation method is simple, economical.The present invention prepares microencapsulated de- cell branch using alginate-polycation
It puts up point, compound liver cell and the building of nonparenchymal cell in vitro culture are used for Biotype artificial liver system three-dimensional liver micro unit, system
Standby simple process, material therefor are easy to get, and cost is relatively low, and exotic material and complicated technology is not used, practical；
2. multiple extracellular matrix components, growth factor and activity point that de- cytoskeleton preferably remains internal liver
Son, immunogenicity is low, the micro- list of liver constructed after compound liver cell, vascular endothelial cell and fibroblast by calcium alginate microsphere
Member more fully hereinafter simulates internal hepatic tissue microenvironment, and with this condition, energy fast and stable obtains liver cell, and further
Improve the activity and function of obtained liver cell；
3. the micro-capsule semi-permeable membrane made of polycation can play effectively immune buffer action, reduces and construct biological people
The immune response of work liver；
4. liver micro unit preparation condition is mild, carry out in physiological conditions, the liver micro unit cell for not influencing building is living
Property, culture scale are easily amplified, and clinical application is conducive to.
Detailed description of the invention
Figure 1A is micro- hepatic tissue unit micrograph of initial preparation of the present invention；B is that two kinds of cells are glimmering in micro- hepatic tissue unit
Light colored graph；
Fig. 2 is the influence result figure adding acellular matrix and secreting to liver cell albumin
Group 1: two-dimentional liver cell, group 2: three-dimensional microencapsulated hepatocyte, group 3: three-dimensional microencapsulated hepatocyte (adds 0.2% to take off carefully
Cytoplasmic matrix ingredient), group 4: three-dimensional microencapsulated hepatocyte+endothelial cell (adding 0.2% acellular matrix ingredient), group 5: three-dimensional micro-capsule
Change liver cell+fibroblast (adding 0.2% acellular matrix ingredient), group 6: three-dimensional microencapsulated hepatocyte+endothelial cell+at fibre
It ties up cell (adding 0.2% acellular matrix ingredient), adds the albumin point of the C3A liver cell of acellular matrix ingredient as the result is shown
Bleeding is flat higher than group is not added with, and many cells co-cultivation group is formed by three-dimensional micro- hepatic tissue and is better than liver cell individually culture group and two
Kind cell co-cultivation group；
Fig. 3 is influence result figure of the cell proportion to three-dimensional micro- hepatic tissue albumin secretion
Three-dimensional liver cell+endothelial cell+fibroblast ratio (each group add the acellular matrix of content 0.2% at
Point): group 1 is 2:1:1, and group 2 is 1:1:1, and group 3 is 1:2:2, and group 4 is 1:3:3, and group 5 is 1:5:5, in co-cultured cell
Skin and fibroblast ratio increase, and the albumin secretion of three-dimensional micro- hepatic tissue increased, the highest when ratio is 1:2:2；
Fig. 4 is influence result figure of the different acellular matrix granule densities to three-dimensional micro- hepatic tissue albumin secretion
It is group 1:0% that liver acellular matrix, which adds concentration (w/v), organizes 2:0.2%, organizes 3:1%, organizes 4:2%, organizes 5:3%,
Group 6:5%, result is group 5 and organizes 6 albumin secretion highest, without significant difference between two groups.
Embodiment 1: many cells co-culture the influence to three-dimensional micro- hepatic tissue function
Miniature pig is anaesthetized, is lost hair or feathers, after disinfection, abdomen hits exactly row cross recess, and hepatic arterial infusion heparin solution is anticoagulant, Men Jing
Arteries and veins be intubated and fix after through peristaltic pump with 37 DEG C of 5mL/min velocity perfusion containing 0.25% trypsase and 0.02%EDTA
Liver is perfused to khaki in PBS solution about 2L, detachment liver week blood vessel, take complete left lateral lobe of liver to freeze in -80 DEG C for 24 hours, with cutting
Lobe of the liver is cut into 3mm thickness 2cm × 2cm size tissue by piece machine, and hepatic tissue piece and de-cell liquid are mixed in 1:15 (w/v) ratio
200 revs/min are placed in bottle, in 4 DEG C of constant-temperature tables.For 24 hours by distilled water, 2% (v/v) Triton X-100 and 0.1% (v/v)
For 24 hours, distilled water 72h successively takes off cell by ammonium hydroxide 96h, 0.1% (w/v) SDS, is by ball mill grinding after the freeze-drying of de- cell tissue piece
10 μm of size particles, sterilize through gamma-ray irradiation.It weighs de- cell granulations and is mixed in the peptide modified quality of RGD, YIGSR, IKVAV
It is in 2% (w/v) sodium alginate soln than 1:1:1 concentration, concentration of the preparation containing de- cytoskeleton particle is 0.2% (w/v's)
Sodium alginate suspension.
Tumor cell of liver C3A, Human umbilical vein endothelial cells HUVEC, human skin fibroblasts HSF are pressed into cell number 1:1:
1 ratio is mixed with above-mentioned suspension, and total cell density is 4 × 106cells/mL.Suspension will be mixed using microcapsules preparing instrument to instill
Calcification 30min in 0.1mol/L calcium chloride solution, addition prepare 0.05% (w/v) Poly-L-Lysine Solution of 10 times of volumes of microballoon
Form a film 10min, is 500 μm with excess charge, acquisition partial size in 0.15% (w/v) sodium alginate soln and encapsulates de- cytoskeleton
The Solid core APA microcapsules of particle and co-cultured cell (see Figure 1A).This microcapsules is placed in addition 0.03mg/ml EGF,
100U/ml penicillin, 100U/ml streptomysin contain in the DMEM in high glucose culture solution of 15% fetal calf serum in 37 DEG C, contain 5%CO2's
Static culture system is cultivated.Individually to cultivate C3A cell, C3A and HUVEC or HSF add the micro- of de- cytoskeleton particle
Capsule culture group compares (see Figure 1B).ELISA detects the secretion of albumin in culture solution supernatant after culture 14 days.Experimental result
See Fig. 2, the albumin secretion level for adding the C3A cell of acellular matrix ingredient as the result is shown, which is higher than, is not added with group, three kinds thin
Born of the same parents' co-cultivation group is formed by three-dimensional micro- hepatic tissue and is better than liver cell individually culture group and two kinds of cell co-cultivation groups.
By tumor cell of liver C3A, Human umbilical vein endothelial cells HUVEC, human skin fibroblasts HSF cell number ratio tune
Whole is 2:1:1,1:1:1,1:2:2, after 1:3:3,1:5:5, still prepares four kinds of different cells encapsulating ratios with former inoculum density
Microcapsules are cultivated 14 days, and ELISA detects the secretion of albumin in culture solution supernatant.Experimental result visible (Fig. 3) is with co-cultivation
Endothelium and fibroblast ratio increase in cell, and the albumin secretion of three-dimensional micro- hepatic tissue is increase accordingly, in 1:2:2 cell ratio
Highest when example.
Influence of 2 acellular matrix of embodiment to three-dimensional micro- hepatic tissue function
Miniature pig is anaesthetized, is lost hair or feathers, after disinfection, abdomen hits exactly row cross recess, and hepatic arterial infusion heparin solution is anticoagulant, Men Jing
Arteries and veins be intubated and fix after through peristaltic pump with 37 DEG C of 5mL/min velocity perfusion containing 0.25% trypsase and 0.02%EDTA
Liver is perfused to khaki in PBS solution about 2L, detachment liver week blood vessel, take complete left lateral lobe of liver to freeze in -80 DEG C for 24 hours, with cutting
Lobe of the liver is cut into 3mm thickness 2cm × 2cm size tissue by piece machine, and hepatic tissue piece and de-cell liquid are mixed in 1:15 (w/v) ratio
200 revs/min are placed in bottle, in 4 DEG C of constant-temperature tables.For 24 hours by distilled water, 2% (v/v) Triton X-100 and 0.1% (v/v)
For 24 hours, distilled water 72h successively takes off cell by ammonium hydroxide 96h, 0.1% (w/v) SDS, is by ball mill grinding after the freeze-drying of de- cell tissue piece
10 μm of size particles, sterilize through gamma-ray irradiation.It is dense that the peptide modified mass ratio 1:1:1 of RGD, YIGSR, IKVAV is dissolved in after weighing
Degree is in 2% (w/v) sodium alginate soln, and the final concentration (w/v) of the de- cytoskeleton particle of acquisition is 0%, 0.2%, 1%,
2%, 3%, 5% suspension.
Tumor cell of liver C3A, Human umbilical vein endothelial cells HUVEC, human skin fibroblasts HSF are pressed into cell number 1:2:
The sodium alginate suspension of 2 ratios and the acellular matrix containing various concentration is mixed with Solid core APA microcapsules culture 2 weeks,
ELISA detects the secretion of albumin in culture solution supernatant.Influence of the different acellular matrix concentration to micro- hepatic tissue function is investigated,
Experimental result is shown in Fig. 4, highest is secreted with the albumin of the 3% and 5% micro- hepatic tissue of acellular matrix addition group in six groups, between two groups
Without significant difference.
To sum up, cell-seeding-density is 4 × 106When a cell/ml, in 3% acellular matrix addition group, C3A:HUVEC:
HSF cell number ratio can get highest albumin secretion under the conditions of being 1:2:2.
Conclusion: by Figure of description and embodiment as it can be seen that the present invention micro- hepatic tissue function obtained better than unicellular or
Two kinds of cell co-cultivation groups and it is not added with acellular matrix group, can be applied to biological artificial liver support system.
Claims (8)
1. a kind of construction method of the micro- hepatic tissue unit of three-dimensional for biological artificial liver support system, it is characterised in that: first
It prepares pig liver and takes off cytoskeleton particle, bracket grain diameter is 10 μm -100 μm, and pig liver obtained is taken off cell branch later
Frame particle is uniformly mixed in sodium alginate soln, and bracket granule density is 0.2%-5% (w/v), is then added into mixed liquor
Enter liver cell, vascular endothelial cell and fibroblast, inoculum density of the total cell in mixed liquor is 1 × 106-1×107A/
ML, wherein liver cell and vascular endothelial cell number proportional region are 5:1-1:3, liver cell and fibroblast number ratio model
It encloses for 5:1-1:3, calcium chloride solution will be instilled containing the mixed liquor of bracket particle and cell later and prepare calcium alginate microsphere, then
Solid core alginate-polylysine microcapsules are made after Poly-L-Lysine Solution film forming is added, add cell culture fluid
In 37 DEG C, contain 5%CO2Static culture system or bioreactor cultivated, pass through cell self assembly realize functionality three
Tie up the building of micro- hepatic tissue unit.
2. according to the method for claim 1, it is characterised in that:
The sodium alginate soln concentration effective range is 2%-4% (w/v)；The molecular weight of sodium alginate is 100-1000kDa,
Guluronic acid and mannuronic acid ratio (G/M) range are 0.2-3:1 in sodium alginate molecule；The sodium alginate is smart ammonia
Acyl-glycyl-aspartic acid (RGD) modification sodium alginate, Tyrosine-Isoleucine-glycine-serine-arginine
(YIGSR) peptide modified sodium alginate, the peptide modified sea Isoleucine-lysine-valine-alanine-valine (IKVAV)
Mosanom is the mixing sodium alginate of 1:1:1 with mass ratio.
3. according to the method for claim 1, it is characterised in that:
The molecular weight of the poly-D-lysine is 2-80kDa, and the Valid concentration of Poly-L-Lysine Solution is 0.05%-
0.2% (w/v)；The ratio of Poly-L-Lysine Solution and the sodium alginate of particle containing acellular matrix suspension is 1:5-1:20 (w/v).
4. according to the method for claim 1, it is characterised in that: the pig liver take off cytoskeleton particle preparation include with
Lower two processes:
Pig liver tissue is sliced preparation process: containing 0.25% (w/v) trypsase and 0.02% (w/v) ethylenediamine tetrem with 37 DEG C
In body liver to khaki, free complete left and right -80 DEG C of siphonal lobe warp freeze the phosphate-buffered perfusion test tube of hepari of sour (EDTA)
>=slice is 1-4mm thick tissue piece afterwards for 24 hours；
The de- cell treatment process of liver organization piece: liver organization piece and de-cell liquid are mixed with 1:15-1:20 (w/v) ratio,
De-cell liquid is followed successively by distilled water for 24 hours, 2% (v/v) Triton X-100 and 0.1% (v/v) ammonia spirit 96h, 0.1% (w/
V) lauryl sodium sulfate (SDS) solution for 24 hours, distilled water 72h, vacuum freeze drying；Cell tissue piece will be taken off after freeze-drying
It is placed in ball milling instrument and is ground into the particle that particle size range is 10 μm -100 μm, frozen after gamma-ray irradiation sterilizes standby in -80 DEG C
With.
5. according to the method for claim 1, it is characterised in that:
The liver cell includes people and mammal source primary hepatocyte, source of human stem cell liver cell, tumorigenic liver cell
One of strain, the unicellular or liver cell aggregation of immortalized hepatocyte strain；The vascular endothelial cell includes people and lactation
One of animal origin Artery, Vein blood endothelial cell strain；The fibroblast includes people and mammal source
One of embryo fibroblast, skin fibroblasts.
6. according to the method for claim 1, it is characterised in that:
The calcium chloride solution concentration is 0.05-0.2mol/L, and calcium chloride solution and the sodium alginate of particle containing acellular matrix are outstanding
The ratio (v/v) of liquid is 1:50-1:200, and the calcification time is 15-30min.
7. according to the method for claim 1, it is characterised in that:
The poly-D-lysine film formation time is 10-20min；The diameter range of the Solid core micro-capsule is 200-2000 μm.
8. according to the method for claim 1, it is characterised in that:
The Biotype artificial liver includes that simple Biotype artificial liver and mixed biology artificial liver support system, institute
The types of bioreactors stated includes stirring-type, filling type, hollow fiber form, gas-lifting type, rotary reactor.
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| 基于脱细胞基质支架材料的肝脏组织工程研究;王立曼;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20150215;E064-93 * |
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