CN105031731B - A kind of quick preparation method for removing cell list leaf liver biological support - Google Patents
A kind of quick preparation method for removing cell list leaf liver biological support Download PDFInfo
- Publication number
- CN105031731B CN105031731B CN201510508929.XA CN201510508929A CN105031731B CN 105031731 B CN105031731 B CN 105031731B CN 201510508929 A CN201510508929 A CN 201510508929A CN 105031731 B CN105031731 B CN 105031731B
- Authority
- CN
- China
- Prior art keywords
- liver
- cell
- biological support
- cell list
- leaf
- 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.)
- Active
Links
Landscapes
- Materials For Medical Uses (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
A kind of quick preparation method for removing cell list leaf liver biological support, without using existing to easily forming thrombus and de-cellular system low the problems such as replanting rate ionic detergent SDS during cellularised again after tissue transplantation, prepared using weakly alkaline Triton infusions method and remove the left outside leaf liver biological support of cell, the addition of NaOH can make the Triton solution of neutrality be in alkalescent in Triton solution, strengthen the effect that cell cracking removes, shorten the time required to removing cell, and employ optimal NaOH with concentration and dosage of Trltion solution, make decellularization best results.
Description
Technical field
The present invention relates to organ-tissue decellularization field, and in particular to a kind of quick single leaf liver acellular organism stent
Preparation method.
Background technology
Extracellular matrix (extracellular matrix, ECM) be filling in intercellular material, the water removal of its component,
Outside electrolyte, a small amount of liquid phase ingredient, mainly also collagen, mucoprotein and proteoglycan etc., they together constitute cell growth
Microenvironment.Liver cell local microenvironment is to include liver cell by the cytokine profiles of extracellular matrix and stroma cell secretion
Growth factor (HGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), epidermal growth factor
Sub (EGF), transforming growth factor (TGF-β) and platelet derived growth factor (PDGF) etc. are formed.Wherein ECM is not only liver
Inner cell growth provides three-D space structure, and the propagation to cell, differentiation and migration have important regulating and controlling effect [1].
Therefore, ECM is the research starting point of timbering material in liver tissue engineering.The appearance for going cell liver technology of preparing is research liver
The biological effect of inner structure type ECM provides a brand-new technology platform, while also provides one for liver tissue engineering
The brand-new biomaterial of kind.It is by physics (freeze thawing), chemistry (SDS) and biology enzyme (trypsase) etc. side to go cell technology
The change [2] that method removes cell without causing ECM components, biological activity and engineering properties from tissue or organ.Naturally
Go cell liver biological support space structure and in liver ECM arrange very similar [3,4]；Remain complete three dimensions knot
Structure, can provide adhesion, growth, differentiation, propagation coherent signal for cell；With good biocompatibility and low immunogenicity
[5,6]；The additionally active cell factor of member-retaining portion such as HGF, therefore be widely used.
Although since 2010 foreign scholar irrigated using different detergents successfully prepare full liver acellular organism stent [4,
7-11], but irrigate path and scheme and lack unified standard, this can be to going the preparation quality and stabilization of cell liver biological support
Property causes to seriously affect.Since liver three-dimensional structure is complicated, the dual blood supply system being made of portal vein and arteria hepatica, and supply
Answer the blood vessels caliber of each lobe of the liver blood flow not quite identical, being irrigated from arteria hepatica OR gate main venous trunk can not be such that each lobe of the liver reaches
Cell effect is gone to consistent.Scholar begins attempt to [12] such as cell list leaf liver, Hussein and uses 0.1% at present
Lauryl sodium sulfate (SDS) prepares pig list leaf liver biology branch by the perfusion of the branch of portal vein of the right outside lobe of the liver of row
Frame.
Preparing both at home and abroad at present goes the detergent of cell liver biological support mainly to have Triton, SDS etc..But SDS conducts
Common detergent, it, which is remained, can cause easily to form thrombus after tissue transplantation, and de-cellular system is during cellularised again
Low the problems such as replanting rate [13].Therefore this research is mainly prepared using weakly alkaline Triton infusions method and removes the left outside leaf of cell
Liver biological support.The addition of NaOH can make the Triton solution of neutrality be in alkalescent, strengthen the effect that its cracking removes cell,
Shorten the time required to removing cell.What is prepared removes the left outside leaf liver cell epimatrix stent of cell through DNA, ELISA, scanning electricity
Mirror, transmission electron microscope, histochemistry, blood vessel casting etc. identify, the results showed that liver inner cell is completely removed substantially, in liver ECM into
Divide the three-D space structure of vascular in (such as FN, LN, IV Collagen Type VI) and liver completely to be retained, complete left outside leaf liver life
The preparation of thing stent.
The content of the invention
The shortcomings that in order to solve the prior art, the present invention is using rat liver as research object, using a kind of Novel fast
Single leaf liver decellularization flow makes acellular organism stent, and detects its morphosis and component, inquires into one kind and goes carefully
New application model of born of the same parents' biological support in liver disease therapy.The present invention provides design and obtain one kind quickly to remove cell
The preparation method of single leaf liver biological support, is used as detergent, preparation is gone by the Triton-100 solution for containing sodium hydroxide
Cell list leaf liver biological support.
A kind of quick preparation method for removing cell list leaf liver biological support, the preparation method comprise the following steps：
(1) in the arteria hepatica 24G indwelling pin intubations of in vitro liver, residual blood in liver is rinsed with heparin PBS solution,
It is in homogeneous khaki to liver；
(2) pylic left outside leaf liver branch and fixation are inserted into trumpet gastric perfusion needle, are held successively at 37 DEG C of constant temperature
Continuous perfusion 400ml contains 1%Trltion X100 2~3h of solution of 0.06%NaOH, and the PBS solution containing penicillin and streptomycin,
Rate of flooding is 3ml/min, and the change of single leaf liver color and form is observed in filling process, treats that liver color becomes semi-transparent
Bright shape, wherein the branch of vascular in liver can be observed, up to single leaf liver biology Acellularized valve, by obtained single leaf liver life
Thing Acellularized valve preserves under the conditions of -80 DEG C.
Perfusion pressure in the step (2) during perfusion is 3.6mmHg.
The PBS solution concentration containing penicillin and streptomycin is 105IU/L in the step (2).
The infusion time of PBS solution of the perfusion containing penicillin and streptomycin is 1h in the step (2).
The beneficial effects of the invention are as follows：The present invention provides a kind of quick preparation side for removing cell list leaf liver biological support
Method, without using existing to easily forming thrombus and de-cellular system low the problems such as replanting rate during cellularised again after tissue transplantation
Ionic detergent SDS, is prepared using weakly alkaline Triton infusions method and removes the left outside leaf liver biological support of cell,
The addition of NaOH can make the Triton solution of neutrality be in alkalescent in Triton solution, the effect that enhancing cell cracking removes, contracting
The time required to short removal cell, and coordinate Triton short with solution min using NaOH, required concentration is low, extracellular matrix
Harmless, this is that other alkaline matters coordinate Triton solution in function not available for liver decellularization, and is employed most
The concentration and dosage with Trltion solution of good NaOH, makes decellularization best results, and the left outside leaf liver of cell is removed in preparation
Extracellular matrix stent is identified through DNA, ELISA, scanning electron microscope, transmission electron microscope, histochemistry, blood vessel casting etc., the results showed that
Hepatic tissue cell can be more fully removed, intactly retains the three-dimensional of ECM components (FN, LN, IV Collagen Type VI etc.) and blood vessel network
Space structure, completes the preparation of left outside leaf liver biological support.
Brief description of the drawings
Fig. 1 is the variation diagram of cell list leaf liver color of the present invention and form；
Fig. 2 is DNA content curve map of the present invention；
Fig. 3 is Elisa quantitative analysis figure of the present invention；
Fig. 4 is scanning electron microscope of the present invention and transmission electron microscope observing figure (A. scanning electron microscope；B. transmission electron microscope, scale show 2 μm)；
Fig. 5 is dyed for hepatic tissue HE of the present invention and PAS colored graphs；
Fig. 6 is liver vessel casting mold figure of the present invention (A. control groups B. goes groups of cells scale to show 5mm)
Embodiment
The acquisition of the full liver of SD rats
Healthy adult SD rat, weighs, and by 0.3~0.4ml/100g10% chloraldurate row intraperitoneal injection of anesthesia, works
After fix, open abdominal cavity, isolate inferior caval vein, the processing of injecting heparin sodium solution row whole body test tube of hepari.Find under liver
The hepatic portal of side, separation arteria hepatica, bile duct and portal vein, detachment bile duct, ligation arteria hepatica, portal vein section start and detachment, at the same time
Detachment vena hepatica, the part that separation liver is connected with diaphram, removes liver, and one group of liver is irrigated decellularization processing, separately
One group of Liver Allograft Preservation in -80 DEG C as a control group.
Go the preparation of cell list leaf liver biological support
(1) one group of Liver Allograft Preservation is not further processed in -80 DEG C and preserved as a control group.
(2) preparation of cell list leaf liver biological support is gone：In vitro liver, arteria hepatica 24G indwelling pin intubations, heparin PBS
Residual blood in liver is rinsed, is in homogeneous khaki to liver.Then pylic left outside leaf liver branch bank gastric perfusion needle is put
Pin, 37 DEG C of constant temperature, irrigates the 1%Trltion X100 solution of (perfusion pressure 3.6mmHg) containing 0.06%NaOH successively
400ml2~3h and PBS solution the 1h containing penicillin and streptomycin (105IU/L).Perfusion liquid is instiled with the speed of 3ml/min, perfusion
During the change of single leaf liver color and form is observed in different time points.Single leaf rami hepatici frame after perfusion is stored in -80 DEG C
Wait to do further identification.
Liver decellularization Data Analysis Services
Gross examination of skeletal muscle：
1. the change of the color and form of each time point list leaf rami hepatici frame after liver and perfusion after blood is gone in observation, such as Fig. 1 institutes
Show：Control group liver is in khaki；The left outside leaf liver of groups of cells color in filling process is gone gradually to bleach and transparence.Finally
Obtained single leaf rami hepatici frame semi-transparent clear, the vascular structure of the visible complete display of naked eyes.
2nd, Genome DNA content is analyzed：
Control group is with going the left outside leaf liver of groups of cells to take 50 1 80mg respectively, through homogenate, cell cracking, removing protein, purifying
Afterwards, genomic DNA (illustrating to operate according to DNA kits) is extracted, ultraviolet specrophotometer measures its concentration.
As a result it is as shown in table 1 below：
DNA content measure (ng/ μ l) in 1 each group list leaf liver of table
Control group and the left outside leaf liver of groups of cells is removed as can be seen from Table 1, row DNA detections, the results show that control group liver
DNA average contents are 4.35 × 103Ng/ μ l, it is 60.35ng/ μ l to remove groups of cells liver dna average content, goes cell proportion can
Up to 98.6%.
DNA content curve map also shows that groups of cells DNA content significantly reduces as seen from Figure 2.
3rd, ELISA quantitative analysis：
Extraction control group and the total protein (being operated according to ELISA kit) for removing the left outside leaf liver of cell.Use microplate reader
The concentration of various kinds of substrates component and cell factor, including Collage I, II, III, IV are detected at 450nm wavelength,
Laminin, fibronectin, HGF, VEFG, TGF-β and EGF.
As shown in Figure 3：Matrix components analysis result shows, go groups of cells Col I, Col II, ColIV, LN and FN it is dense
Degree is compared with control group, no difference of science of statistics (Fig. 3 A).Cytokine analysis the result shows that, go groups of cells HGF, TGF-β,
The concentration of VEGF, EGF are similar to control group, VEGF P ＜ 0.05, statistically significant (Fig. 3 B).
4. scanning electron microscope and transmission electron microscope observing：
Control group and the left outside leaf liver of groups of cells is removed, be cut into the tissue block of 1.0mm3 sizes, 4 DEG C of 2.5% glutaraldehyde respectively
It is fixed to stay overnight.After PBS is rinsed, control group is taken to be fixed with going groups of cells portion of tissue block to carry out osmic acid, the dyeing of acetic acid uranium, acetone
Serial dehydration, epoxy resin embedding agent are impregnated with, embedding, are made ultra-thin section, are observed under transmission electron microscope；Remaining tissue block is into serving a round of liquor to the guests
Smart serial dehydration, is dried, plated film, is observed under scanning electron microscope.
Go lobuli hepatis contour structure in the left outside leaf rami hepatici frame of groups of cells substantially complete as shown in figure 4, scanning electron microscope is visible, carefully
Extracellular matrix is in continuous " fishing net shaped " structure, and tube chamber is complete, hollow and acellular residual (Fig. 4 A).Transmission electron microscope observing is shown in stent
The extracellular matrix components such as interior collagenous fibres, the extracellular matrix after cell removes are presented the cell outline collapsed, have no cell
(Fig. 4 B).
5.HE is dyed and PAS dyeing observations：
Control group and the left outside leaf liver of groups of cells is removed, fixed through 4% paraformaldehyde, alcohol serial dehydration, dimethylbenzene is transparent,
Light Microscopic observation after waxdip, embedding, section, conventional H E dyeing and PAS dyeing.
As shown in figure 5, HE dyeing observation control group liver organization structures understand that nucleus is clear (Fig. 5 A)；Go groups of cells
The only visible extracellular matrix components being arranged radially, they completely connect and accumulate central vein, liver to center each other
Cell residue (Fig. 5 B) is not found in sinus.PAS dyeing observations go groups of cells liver glycogen component to retain complete (Fig. 5 C).
6. liver vessel casting mold is observed
Control group and the left outside leaf liver of groups of cells is removed, with constant pressure in hepatic arterial infusion red polymethyl methacrylate
(PMMA) 0.5~1ml of solution, in portal vein perfusion blueness PMMA 1~2ml of solution, in vena hepatica perfusion yellow PMMA solution 2~
3ml, natural cooling.50% hcl corrosion of control group liver 5~7 days, removes groups of cells liver 1~3 day, clear water is rinsed well, body
The form of blood vessel and distribution in left outside leaf liver are observed under stereomicroscope.
As shown in fig. 6, according to group and going groups of cells liver vessel to be distributed in " branch sample ", control group liver vessel is closeer
Collection, form are complete；Go blood vessel in groups of cells liver sparse, but vessel branch is still high-visible, tiny lateral configuration has retained
Whole (Fig. 6).
Claims (4)
1. a kind of quick preparation method for removing cell list leaf liver biological support, it is characterised in that the preparation method includes
Following steps：
(1) in the arteria hepatica 24G indwelling pin intubations of in vitro liver, residual blood in liver is rinsed with heparin PBS solution, to liver
Dirty is in homogeneous khaki；
(2) pylic left outside leaf liver branch and fixation are inserted into trumpet gastric perfusion needle, are persistently filled successively at 37 DEG C of constant temperature
Note 400ml contains 1%Trltion X100 2~3h of solution of 0.06%NaOH, and the PBS solution containing penicillin and streptomycin, perfusion
Speed is 3ml/min, and the change of single leaf liver color and form is observed in filling process, treats that liver color becomes translucent
Shape, wherein the branch of vascular in liver can be observed, up to single leaf liver biology Acellularized valve, by obtained single leaf liver biology
Acellularized valve preserves under the conditions of -80 DEG C.
A kind of 2. quick preparation method for removing cell list leaf liver biological support according to claim 1, it is characterised in that
Perfusion pressure in the step (2) during perfusion is 3.6mmHg.
A kind of 3. quick preparation method for removing cell list leaf liver biological support according to claim 1, it is characterised in that
The PBS solution concentration containing penicillin and streptomycin is 105 IU/L in the step (2).
A kind of 4. quick preparation method for removing cell list leaf liver biological support according to claim 1, it is characterised in that
The infusion time of PBS solution of the perfusion containing penicillin and streptomycin is 1h in the step (2).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510508929.XA CN105031731B (en) | 2015-08-18 | 2015-08-18 | A kind of quick preparation method for removing cell list leaf liver biological support |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510508929.XA CN105031731B (en) | 2015-08-18 | 2015-08-18 | A kind of quick preparation method for removing cell list leaf liver biological support |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105031731A CN105031731A (en) | 2015-11-11 |
| CN105031731B true CN105031731B (en) | 2018-04-17 |
Family
ID=54439083
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510508929.XA Active CN105031731B (en) | 2015-08-18 | 2015-08-18 | A kind of quick preparation method for removing cell list leaf liver biological support |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105031731B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107456604B (en) * | 2017-06-27 | 2018-05-18 | 温州医科大学附属第一医院 | It can be applied to part pancreas decellularization biological support, the preparation method and applications of Xun Huan docking transplanting |
| CN112957534A (en) * | 2021-04-06 | 2021-06-15 | 海南大学 | Preparation method of acellular dermal matrix material |
| CN114712562A (en) * | 2022-04-08 | 2022-07-08 | 上海亚朋生物技术有限公司 | Preparation process of acellular freeze-dried amnion product |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101376013B1 (en) * | 2005-08-26 | 2014-03-19 | 리전츠 오브 더 유니버스티 오브 미네소타 | Decellularization and recellularization of organs and tissues |
| WO2007124127A2 (en) * | 2006-04-21 | 2007-11-01 | Wake Forest University Health Sciences | Structurally modified acellular tissue engineering scaffolds and methods of production |
| CN101564554A (en) * | 2009-06-03 | 2009-10-28 | 南方医科大学珠江医院 | Kit and method used for obtaining full liver stent |
| CN104208751A (en) * | 2014-08-19 | 2014-12-17 | 温州医科大学 | Preparation method for novel kidney acellularized biological scaffold |
-
2015
- 2015-08-18 CN CN201510508929.XA patent/CN105031731B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN105031731A (en) | 2015-11-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Choi et al. | Decellularized extracellular matrix derived from porcine adipose tissue as a xenogeneic biomaterial for tissue engineering | |
| US20200164109A1 (en) | Methods of producing multi-layered tubular tissue constructs | |
| WO2016138701A1 (en) | Artificial blood vessel and preparation method thereof | |
| CN106474549B (en) | The novel tissue tissue-engineered nerve of MicroRNA gene mediated constructs and its in the application for repairing neurologic defect | |
| CN105188786B (en) | The purposes of particulate and endothelial cell and the organ of acellular and tissue | |
| ES2677945T3 (en) | Production of artificial tissues by tissue engineering using fibrin and agarose biomaterials | |
| CN1997410A (en) | Cell-independent fabrication of tissue equivalents | |
| CN105031731B (en) | A kind of quick preparation method for removing cell list leaf liver biological support | |
| CN106729984A (en) | A kind of Isin glue collagen repairs sponge and preparation method thereof | |
| CN110772669A (en) | Biological ink for 3D printing of artificial skin | |
| US20180042970A1 (en) | Injectable Microtissue Systems, Devices, and Methods | |
| Ajalloueian et al. | One-stage tissue engineering of bladder wall patches for an easy-to-use approach at the surgical table | |
| CN104292497B (en) | A kind of preparation method of recombination human source collagen bio-sponge | |
| CN104822875A (en) | Product Made of Silk | |
| CN114854677B (en) | Microfluidic bionic fiber for cell culture meat production and preparation method and application thereof | |
| CN109529121A (en) | A kind of Acellular trachea matrix and preparation method thereof | |
| CN104399125A (en) | Method for differentiating epidermal stem cells to sweat gland-like epithelial cells | |
| CN111544648A (en) | Protein-modified PLGA microspheres and tissue-engineered nerves constructed by same | |
| CN110585480B (en) | Forming method of self-supporting silk fibroin catheter stent | |
| Mahdavi et al. | Development and in vitro evaluation of photocurable GelMA/PEGDA hybrid hydrogel for corneal stromal cells delivery | |
| CN108236739A (en) | A kind of advanced composite material (ACM) applied to cartilage damage reparation | |
| CN102697581B (en) | Method for constructing tissue engineering blood vessel | |
| CN110464877A (en) | A kind of preparation method and its effect evaluation method of acellular nerve allografts | |
| CN110218344A (en) | A kind of elastic hydrogel fiber and its preparation method and application | |
| Liu et al. | Preparation of rat whole-kidney acellular matrix via peristaltic pump |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |