CN103525753B - A kind of separation method of endothelium Clone formation cell - Google Patents
A kind of separation method of endothelium Clone formation cell Download PDFInfo
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Abstract
The invention discloses a kind of separation method of endothelium Clone formation cell, comprise the steps: (1) enzymic digestion: get umbilical cord, umbilical vein is rinsed to the liquid-transparent flowed out with alkalescence or neutral buffered liquid, the mixed enzyme of collagenase or collagenase and pancreatin is filled with in umbilical vein, digestion 15 ~ 60min, collects Digestive system; (2) mechanical scraping: cut open by umbilical vein, exposes umbilical vein internal surface, with cell scraping umbilical vein internal surface 3 ~ 10 minutes, with alkalescence or the washing of neutral buffered liquid, collects washings; (3) washings of the Digestive system of step (1) and step (2) is merged, centrifugal, remove supernatant.The separation method of endothelium Clone formation cell of the present invention, can effectively be separated endothelium Clone formation cell from umbilical vein, potential applicability in clinical practice is excellent.
Description
Technical field
The present invention relates to the separation method of endothelium Clone formation cell.
Background technology
Endothelial progenitor cells is the precursor cell of endotheliocyte, in maintenance blood vessel endothelium ambient stable, play important role.According to endothelial progenitor cells (EPCs) colony morphology, time of occurrence successively and cellular proliferative potential, the peripheral blood MNCs of vitro culture can differentiate EPCs in early stage and late period.Endothelium Clone formation cell is the EPCs subgroup in a kind of late period of discovered in recent years, because having stronger multiplication capacity and vascularization ability, is desirable transplanting seed cell.Endothelium Clone formation cell can be separated and obtain in the middle of the Various Tissues such as marrow, Cord blood, peripheral blood, umbilical cord.
Umbilical vein contains a large amount of endothelium Clone formation cells, draws materials easily, simple to operate, is the good source being separated endothelium Clone formation cell.At present, usually take enzyme digestion or machinery to scrape and follow the example of, obtain endothelium clone cell from umbilical vein, be inoculated in applicable substratum and cultivate.But adopt the method for mechanical scraping, endothelium Clone formation cell can not successfully be separated with collagenous tissue, and separation efficiency is low, and the separation of enzyme digestion is also often thorough not, and separation efficiency is low.
Set up a kind of endothelium Clone formation cellular segregation cultural method efficiently, have important meaning to clinical large-scale application.
Summary of the invention
In order to overcome the defect of prior art, the invention provides a kind of separation method of new endothelium Clone formation cell.
The separation method of endothelium Clone formation cell of the present invention, comprises the steps:
(1) enzymic digestion: get umbilical vein, rinsing the liquid-transparent to flowing out with alkalescence or neutral buffered perfusion, pouring into the mixed enzyme of collagenase or collagenase and pancreatin, close two ends, and digestion 15 ~ 60min, opens two ends, collects Digestive system;
(2) mechanical scraping: cut open by umbilical vein, exposes umbilical vein internal surface, with cell scraping umbilical vein internal surface 3 ~ 10 minutes, then with alkalescence or the washing of neutral buffered liquid, collects washings;
(3) washings of the Digestive system of step (1) and step (2) is merged, centrifugal, remove supernatant.
In step (1), described alkalescence or neutral buffered liquid are PBS damping fluid, DPBS damping fluid or Hanks damping fluid.
In step (1), described collagenase is II Collagenase Type.
In step (1), the concentration of described collagenase is 0.2%, and the concentration of described pancreatin is 0.5%, and the volume ratio of collagenase and pancreatin is 1:1.
In step (1), the temperature of described digestion is 37 DEG C.
In step (1), the time of described digestion is 15 ~ 30min.
In step (2), the time of described scraping is 5 minutes.
In step (2), described alkalescence or neutral buffered liquid are PBS damping fluid, DPBS damping fluid or Hanks damping fluid.
In step (2), the consumption of described damping fluid is 0.5 ~ 1.5 times (ml/cm) of umbilical vein length.Preferably, the consumption of described damping fluid is 1 times (ml/cm) of umbilical vein length.
The separation method of endothelium Clone formation cell of the present invention, scraped the particular combination of taking technique by enzymic digestion technology and machinery, be effectively separated from umbilical vein tissue by endothelium Clone formation cell, the cell quantity of acquisition is many, separation efficiency is high, and potential applicability in clinical practice is excellent.
Below by embodiment, the present invention is described in further details, but be not limitation of the present invention, according to foregoing of the present invention, according to ordinary technical knowledge and the customary means of this area, not departing under the present invention's above-mentioned basic fundamental thought prerequisite, the amendment of other various ways, replacement or change can also be made.
Accompanying drawing illustrates:
Fig. 1 is that P0 is for cell morphological characteristic;
Fig. 2 is endothelium Clone formation cells cell surface Mark Detection;
Fig. 3 is the LDL endocytosis experiment of endothelium Clone formation cell, and the optical signal in figure is the positive signal that the LDL after endothelium Clone formation cell endocytic sends under the exciting of laser;
Fig. 4 is that the extracorporeal blood vessel of endothelium Clone formation cell forms experiment.
Embodiment:
The separation method of embodiment 1 endothelium Clone formation of the present invention cell
Umbilical cord takes from full-term normal delivery or caesarean healthy puerpera, is put in containing 4 DEG C of preservations in dual anti-PBS solution after collection, process in 48h.Repeatedly clean, and with phosphoric acid buffer PBS irrigation umbilical vein, until flowing liquid is transparent.
Clamp umbilical cord one end with mosquito forceps, with liquid-transfering gun, II Collagenase Type (concentration 0.2%) added and is full of umbilical vein, clamping the umbilical cord the other end with mosquito forceps; 37 DEG C of shaking tables digest 30 minutes; After having digested, take off mosquito forceps, collect Digestive system in 50ml centrifuge tube; Umbilical cord is cut open along umbilical vein, exposes umbilical vein internal surface, scrape with cell and gently scrape umbilical vein 5 minutes; With PBS solution cleaning, the consumption of described damping fluid is 1 times (ml/cm) of umbilical vein length, collects in washings and centrifuge tube.
By the centrifuge tube of collecting cell 4 DEG C, under the condition of 500g, centrifugal 5 minutes.
The separation method of embodiment 2 endothelium Clone formation of the present invention cell
Umbilical cord takes from full-term normal delivery or caesarean healthy puerpera, is put in containing 4 DEG C of preservations in dual anti-DPBS damping fluid after collection, process in 48h.Repeatedly clean, and with phosphoric acid buffer PBS irrigation umbilical vein, until flowing liquid is transparent.
Clamp umbilical cord one end with mosquito forceps, with liquid-transfering gun, II Collagenase Type (concentration 0.2%) added and is full of umbilical vein, clamping the umbilical cord the other end with mosquito forceps; 37 DEG C of shaking tables disappear 15 minutes; After having digested, take off mosquito forceps, collect Digestive system in 50ml centrifuge tube; Umbilical cord is cut open along umbilical vein, exposes umbilical vein internal surface, scrape with cell and gently scrape umbilical vein 3 minutes; Use DPBS buffer solution for cleaning, the consumption of described damping fluid is 1.5 times (ml/cm) of umbilical vein length, collects in washings and centrifuge tube.
By the centrifuge tube of collecting cell 4 DEG C, under the condition of 500g, centrifugal 5 minutes.
The separation method of embodiment 3 endothelium Clone formation of the present invention cell
Umbilical cord takes from full-term normal delivery or caesarean healthy puerpera, is put in containing 4 DEG C of preservations in dual anti-Hanks damping fluid after collection, process in 48h.Repeatedly clean, and with phosphoric acid buffer PBS irrigation umbilical vein, until flowing liquid is transparent.
Clamp umbilical cord one end with mosquito forceps, with liquid-transfering gun by II Collagenase Type (concentration 0.2%) and pancreatin (concentration is 0.5%) by 1:1(v/v) mixed enzyme (two enzyme) that forms, add and be full of umbilical vein, clamping the umbilical cord the other end with mosquito forceps; 37 DEG C of shaking tables disappear 60 minutes; After having digested, take off mosquito forceps, collect Digestive system in 50ml centrifuge tube; Umbilical cord is cut open along umbilical vein, exposes umbilical vein internal surface, scrape with cell and gently scrape umbilical vein 10 minutes; Use Hanks buffer solution for cleaning, the consumption of described damping fluid is 0.5 times (ml/cm) of umbilical vein length, collects in washings and centrifuge tube.
By the centrifuge tube of collecting cell 4 DEG C, under the condition of 500g, centrifugal 5 minutes.
The comparison of the separation method of embodiment 4 different endothelium Clone formation cell
1, materials and methods
Umbilical cord takes from full-term normal delivery or caesarean healthy puerpera, is put in containing 4 DEG C of preservations in dual anti-PBS solution after collection, process in 48h.Repeatedly clean, and with phosphoric acid buffer PBS irrigation umbilical vein, until flowing liquid is transparent.Be separated with diverse ways respectively.
A. enzyme digestion: clamp umbilical cord one end with mosquito forceps, adds with liquid-transfering gun II Collagenase Type (0.2%) and is full of umbilical vein, clamping the umbilical cord the other end with mosquito forceps; 37 DEG C of shaking tables digest 15 minutes; After having digested, take off mosquito forceps, collect Digestive system in 50ml centrifuge tube, then rinse vein by PBS solution, the consumption of PBS solution is 1 times (ml/cm) of umbilical vein length, collects washings to centrifuge tube.
B. machinery is scraped and is followed the example of: cut open along umbilical vein by umbilical cord, exposes umbilical vein internal surface, scrapes umbilical vein 5 minutes with cell; With PBS solution cleaning, the consumption of PBS solution is 1 times (ml/cm) of umbilical vein length, collects washings in centrifuge tube.
C. the inventive method (enzymic digestion+machinery is scraped and followed the example of): clamp umbilical cord one end with mosquito forceps, with liquid-transfering gun, II Collagenase Type (0.2%) added and is full of umbilical vein, clamping the umbilical cord the other end with mosquito forceps; 37 DEG C of shaking tables digest 30 minutes; After having digested, take off mosquito forceps, collect Digestive system in 50ml centrifuge tube; Umbilical cord is cut open along umbilical vein, exposes umbilical vein internal surface, scrape umbilical vein 5 minutes with cell; With PBS cleaning, the consumption of PBS solution is 1 times (ml/cm) of umbilical vein length, collects in washings and centrifuge tube.
After three kinds of method process, the centrifuge tube of collecting cell 4 DEG C respectively, 500g, centrifugal 5 minutes.Use EGM2-MV substratum resuspended respectively, be inoculated in the 35mm culture dish after I type Collagen type-I bed board, inoculate the amount inoculating cell of a 35mm ware by every 2cm umbilical cord, be placed in 37 DEG C, the CO of volume fraction 5%
2cultivate in saturated humidity incubator.According to cell growth status, every 2-3 days full dose changes liquid once.When the cell of the fastest group of growth reaches 90% degree of converging, the pancreatin with 0.05%/EDTA digestion, count ratio respectively organizes the quantity of P0 for cell.
2, experimental result
As shown in Figure 1, the cellular form that the inventive method obtains is paving stone shape, and refractive power is better, belongs to endothelium Clone formation cell.
The endothelium Clone formation cell P0 algebraic quantity of three kinds of method acquisitions is as shown in table 1:
The cell quantity that table 1 different methods obtains compares
| P0 is for harvest yield (ten thousand) | |
| Machinery scraping | 8.5 |
| Collagenase digesting | 13.8 |
| Collagenase digesting+mechanical scraping | 31.5 |
As shown in table 1, machinery is scraped the P0 followed the example of with enzyme digestion acquisition and is only respectively 8.5 ten thousand and 13.8 ten thousand for cell, and the technique that method of the present invention adopts enzyme digestion and mechanical scraping to combine, obtaining P0 for cell is 31.5 ten thousand, is 3.7 times and 2.3 times that adopt separately the first two kind method.
The endothelium Clone formation cell quantity that the inventive method obtains, highly significant scrapes the endothelium Clone formation cell quantity of following the example of and obtaining with enzyme digestion higher than being used alone machinery, illustrating that the present invention combines with enzyme digestion by being scraped by machinery to follow the example of, very substantial increasing the separation efficiency of endothelium Clone formation cell.
The optimization of embodiment 5 endothelium Clone formation of the present invention cell isolation method
1, experimental technique
All the other conditions are with embodiment 1, and digestive ferment is respectively 0.2%II Collagenase Type (single enzyme), or 0.2%II Collagenase Type mixes with 0.5% pancreatin the two enzymes formed by 1:1; Digestion time is respectively 15 minutes, 30 minutes and 60 minutes.
When the cell of the fastest group of growth reaches 90% degree of converging, the pancreatin with 0.05%/EDTA digestion, count ratio respectively organizes the quantity of P0 for cell.
2, experimental result
After the digestion of different enzymic digestion conditions, then scraping collecting cell.After two enzymic digestion, obtain more cell (table 2) through cultivating.
Table 2 adopts the method for collagenase digesting+mechanical scraping, to the result that different enzymic digestion conditions is optimized
| 15min30min60min collagenase 11.8 ten thousand 24.0 10,000 3.3 ten thousand pancreatin+collagenase 31.5 ten thousand 24.8 10,000 2.8 ten thousand |
As shown in table 2, when adopting separately the digestion of II Collagenase Type, the cell quantity of acquisition, along with the prolongation of digestion time, first increases and reduces afterwards, and during digestion 30min, the cell quantity of acquisition is maximum, is 24.0 ten thousand; During mixed enzyme digestion with pancreatin and II Collagenase Type, the cell quantity of acquisition reduces along with the prolongation of digestion time, and during digestion 15mim, the cell quantity of acquisition is maximum, is 31.5 ten thousand, is better than adopting separately II Collagenase Type to digest the cell quantity obtained.
Experimental result illustrates, adopt the mixed enzyme digestion 15min of pancreatin and II Collagenase Type, the endothelium Clone formation cell quantity of acquisition is maximum.
The qualification of the endothelium Clone formation cell that embodiment 6 the inventive method is separated
1, experimental technique
(1) Secondary Culture of endothelium Clone formation cell
Example 3 the inventive method obtain P0 for cell, by 10000/cm
2by density carry out inoculation of going down to posterity.In Secondary Culture process, within every 3 days, full dose changes liquid, until when attached cell reaches 90% degree of converging, repeats aforesaid operations and goes down to posterity.
(2) endothelium Clone formation cell surface marker detects
Enzymic digestion+machinery scrapes the P5 that follows the example of separation and Culture for after cell routine digestion, and with CD14, CD73, VEGFR2 of PE mark, CD31, CD90, CD34, CD45 antibody of FITC mark and corresponding Isotype control labeled cell thereof, carry out flow cytometer detection.
(3) endothelium Clone formation cell low-density lipoprotein (LDL) endocytosis experiment
Enzymic digestion+machinery scrapes the P5 following the example of separation and Culture when reaching 70% for Growth of Cells to degree of converging, and adds the LDL that 10 μ g/ml Dil-Ac mark, hatches 4 hours for 37 DEG C; Remove substratum, PBS washs 3 times, fixes 30 minutes with 3% paraformaldehyde in room temperature; 3 times are repeatedly cleaned, at fluorescence microscopy Microscopic observation with PBS.
(4) endothelium Clone formation cells in vitro vascularization experiment
Matrigel is placed on ice, thawing of spending the night; Matrigel after melting is added in 24 orifice plates of precooling, hatches 1 hour for 37 DEG C; By P5 for the pancreatin/EDTA digestion of 0.05% of endothelium Clone formation cell, be inoculated in ready Matrigel culture plate by the amount of 10000 cell per well; Be placed in 37 DEG C, cultivate after 4 hours in the CO2 saturated humidity incubator of volume fraction 5%, take out and examine under a microscope tubular structure formational situation.
2, experimental result
Experimental result is as shown in Figure 2 to 4:
As shown in Figure 2, cell surface expression CD31, VEGFR2 and CD73 mark of the endothelium Clone formation cell in P5 generation; Do not express CD14, CD90, CD45, and CD34 is the expression of diffuse type, the feature that the surface marker meeting the endothelium Clone formation cell of bibliographical information is expressed.
As shown in Figure 3, after hatching with the LDL after mark, carry out endocytosis to the latter, under the exciting of laser, present fluorescent signal, showed cell has normal LDL endocytosis.
As shown in Figure 4, in Matrigel culture surface, form tubular structure, and be interconnected, formed netted, show cell and there is very strong extracorporeal blood vessel Forming ability.
Experimental result illustrates, the present invention is separated the cell obtained, and has the surface marker of endothelium Clone formation cell, has LDL endocytosis and very strong extracorporeal blood vessel Forming ability, belongs to endothelium Clone formation cell.
Experimental result illustrates, the inventive method can from umbilical vein, and be effectively separated endothelium Clone formation cell, the cell quantity of acquisition is many, and application prospect is good.
Claims (7)
1. a separation method for endothelium Clone formation cell, is characterized in that: comprise the steps:
(1) enzymic digestion: get umbilical vein, rinsing the liquid-transparent to flowing out with alkalescence or neutral buffered perfusion, pouring into the mixed enzyme of collagenase or collagenase and pancreatin, close two ends, and digestion 15 ~ 60min, opens two ends, collects Digestive system;
(2) mechanical scraping: cut open by umbilical vein, exposes umbilical vein internal surface, with cell scraping umbilical vein internal surface 3 ~ 10 minutes, then with alkalescence or the washing of neutral buffered liquid, collects washings;
(3) washings of the Digestive system of step (1) and step (2) is merged, centrifugal, remove supernatant;
In step (1), described collagenase is II Collagenase Type;
In step (1), the concentration of described collagenase is 0.2%, and the concentration of described pancreatin is 0.5%, and the volume ratio of collagenase and pancreatin is 1:1;
In step (1), the temperature of described digestion is 37 DEG C.
2. separation method according to claim 1, is characterized in that: in step (1), and described alkalescence or neutral buffered liquid are PBS damping fluid, DPBS damping fluid or Hanks damping fluid.
3. separation method according to claim 1, is characterized in that: in step (1), and the time of described digestion is 15 ~ 30min.
4. separation method according to claim 1, is characterized in that: in step (2), and the time of described scraping is 5 minutes.
5. separation method according to claim 1, is characterized in that: in step (2), and described alkalescence or neutral buffered liquid are PBS damping fluid, DPBS damping fluid or Hanks damping fluid.
6. separation method according to claim 1, is characterized in that: in step (2), every centimetre of umbilical vein 0.5 ~ 1.5ml buffer solution.
7. separation method according to claim 6, is characterized in that: every centimetre of umbilical vein 1ml buffer solution.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1314440A2 (en) * | 2001-11-22 | 2003-05-28 | Nipro Corporation | Cultured skin and method of maufacturing the same |
| CN101199550A (en) * | 2007-11-16 | 2008-06-18 | 中国医学科学院血液学研究所 | Method for preparing preparation containing endothelial progenitor cells using umbilical cord or placenta and use thereof |
| CN101353644A (en) * | 2008-09-12 | 2009-01-28 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of vascular endothelial cell and its preparation method and application |
| CN102827805A (en) * | 2012-09-25 | 2012-12-19 | 江苏省农业科学院 | HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method |
| CN103215218A (en) * | 2012-01-21 | 2013-07-24 | 中国人民解放军军事医学科学院附属医院 | Method for separating and culturing endothelial clone formative cell |
-
2013
- 2013-09-23 CN CN201310436011.XA patent/CN103525753B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1314440A2 (en) * | 2001-11-22 | 2003-05-28 | Nipro Corporation | Cultured skin and method of maufacturing the same |
| CN101199550A (en) * | 2007-11-16 | 2008-06-18 | 中国医学科学院血液学研究所 | Method for preparing preparation containing endothelial progenitor cells using umbilical cord or placenta and use thereof |
| CN101353644A (en) * | 2008-09-12 | 2009-01-28 | 中国人民解放军军事医学科学院基础医学研究所 | A kind of vascular endothelial cell and its preparation method and application |
| CN103215218A (en) * | 2012-01-21 | 2013-07-24 | 中国人民解放军军事医学科学院附属医院 | Method for separating and culturing endothelial clone formative cell |
| CN102827805A (en) * | 2012-09-25 | 2012-12-19 | 江苏省农业科学院 | HUVEC (human umbilical vein endothelial cell) separation, culture and subculture method |
Non-Patent Citations (1)
| Title |
|---|
| "胶原酶脐带灌注消化法在人脐带静脉内皮细胞的原代分离培养中的作用";谢明斌 等;《山东医药》;20091231;第49卷(第51期);第10页右栏第2段-第11页左栏第3段 * |
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