CN103525753A - Method for separating endothelial colony-forming cells - Google Patents
Method for separating endothelial colony-forming cells Download PDFInfo
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- CN103525753A CN103525753A CN201310436011.XA CN201310436011A CN103525753A CN 103525753 A CN103525753 A CN 103525753A CN 201310436011 A CN201310436011 A CN 201310436011A CN 103525753 A CN103525753 A CN 103525753A
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Abstract
The invention discloses a method for separating endothelial colony-forming cells. The method comprises the following steps: (1) enzymatic digestion: taking an umbilical cord, washing an umbilical cord vein with an alkaline or neutral buffer solution till effluent liquid is transparent, filling collagenase or a mixed enzyme of collagenase and trypsin into the umbilical cord vein, digesting for 15-60min, and collecting a digestion solution; (2) mechanical scraping: dissecting the umbilical cord vein, exposing the inner surface of the umbilical cord vein, scraping the inner surface of the umbilical cord vein with a cell scraper for 3-10min, washing with the alkaline or neutral buffer solution, and collecting a washing solution; (3) merging the digestion solution in the step (1) and the washing solution in the step (2), centrifuging, and removing supernatant liquid for preparation. By adopting the method for separating the endothelial colony-forming cells, disclosed by the invention, the endothelial colony-forming cells can be effectively separated from the umbilical cord vein, and clinical application prospects are excellent.
Description
Technical field
The present invention relates to the separation method that interior picalon forms cell.
Background technology
Endothelial progenitor cells is the precursor cell of endotheliocyte, is playing the part of important role in maintaining blood vessel endothelium ambient stable.According to endothelial progenitor cells (EPCs) clone form, time of occurrence successively and cell proliferation potentiality, the peripheral blood MNCs of vitro culture can differentiate EPCs in early stage and late period.Interior picalon forms the EPCs subgroup in a kind of late period that cell is discovered in recent years, because having stronger multiplication capacity and vascularization ability, is desirable transplanting seed cell.Interior picalon forms cell can be from separated acquisition in the middle of the Various Tissues such as marrow, Cord blood, peripheral blood, umbilical cord.
Umbilical vein contains a large amount of interior picalons and forms cell, draws materials easily, simple to operate, is the good source that separated interior picalon forms cell.At present, conventionally take enzyme digestion or machinery to scrape and follow the example of, from umbilical vein, obtain endothelium clone cell, be inoculated in applicable substratum and cultivate.But, adopting the method for mechanical scraping, interior picalon forms cell can not be successfully separated with collagenous tissue, and separation efficiency is low, and the separation of enzyme digestion is also often thorough not, separation efficiency is low.
Set up a kind of efficiently interior picalon and form cellular segregation cultural method, clinical large-scale application is had to important meaning.
Summary of the invention
In order to overcome the defect of prior art, the invention provides the separation method that a kind of new interior picalon forms cell.
In the present invention, picalon forms the separation method of cell, comprises the steps:
(1) enzymic digestion: get umbilical vein, rinse to the liquid-transparent flowing out with alkalescence or neutral buffered perfusion, pour into the mixed enzyme of collagenase or collagenase and pancreatin, sealing two ends, digest 15～60min, open two ends, collect Digestive system;
(2) mechanical scraping: umbilical vein is cut open, exposed umbilical vein internal surface, with cell scraping umbilical vein internal surface 3～10 minutes, then with alkalescence or the washing of neutral buffered liquid, collect 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 ℃.
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.
In the present invention, picalon forms the separation method of cell, the particular combination of scraping taking technique by enzymic digestion technique and machinery, forms interior picalon cell separated from umbilical vein tissue effectively, and the cell quantity of acquisition is many, separation efficiency is high, and potential applicability in clinical practice is good.
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 skill knowledge and the customary means of this area, not departing under the above-mentioned basic fundamental thought of the present invention prerequisite, can also make modification, replacement or the change of other various ways.
Accompanying drawing explanation:
Fig. 1 is that P0 is for cell morphological characteristic;
Fig. 2 is that interior picalon forms the detection of cell surface marker;
Fig. 3 is the LDL endocytosis experiment that interior picalon forms cell, and the optical signal in figure is that interior picalon forms the positive signal that the LDL after cell endocytic sends under the exciting of laser;
Fig. 4 is the extracorporeal blood vessel formation experiment that interior picalon forms cell.
Embodiment:
In embodiment 1 the present invention, picalon forms the separation method of cell
Umbilical cord is taken from full-term normal delivery or caesarean healthy puerpera, is put in containing 4 ℃ of preservations in dual anti-PBS solution after collection, in 48h, processes.Repeatedly clean, and with phosphoric acid buffer PBS irrigation umbilical vein, until flowing liquid is transparent.
With mosquito forceps, clamp umbilical cord one end, with liquid-transfering gun, II Collagenase Type (concentration 0.2%) is added and is full of umbilical vein, with mosquito forceps, clamp the umbilical cord the other end; 37 ℃ 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, exposed umbilical vein internal surface, with cell, scrape and gently scrape umbilical vein 5 minutes; With PBS solution, clean, 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 under 4 ℃, the condition of 500g, centrifugal 5 minutes.
In embodiment 2 the present invention, picalon forms the separation method of cell
Umbilical cord is taken from full-term normal delivery or caesarean healthy puerpera, is put in containing 4 ℃ of preservations in dual anti-DPBS damping fluid after collection, in 48h, processes.Repeatedly clean, and with phosphoric acid buffer PBS irrigation umbilical vein, until flowing liquid is transparent.
With mosquito forceps, clamp umbilical cord one end, with liquid-transfering gun, II Collagenase Type (concentration 0.2%) is added and is full of umbilical vein, with mosquito forceps, clamp the umbilical cord the other end; 37 ℃ 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, exposed umbilical vein internal surface, with cell, scrape 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 under 4 ℃, the condition of 500g, centrifugal 5 minutes.
In embodiment 3 the present invention, picalon forms the separation method of cell
Umbilical cord is taken from full-term normal delivery or caesarean healthy puerpera, is put in containing 4 ℃ of preservations in dual anti-Hanks damping fluid after collection, in 48h, processes.Repeatedly clean, and with phosphoric acid buffer PBS irrigation umbilical vein, until flowing liquid is transparent.
With mosquito forceps, clamp umbilical cord one end, with liquid-transfering gun, II Collagenase Type (concentration 0.2%) and pancreatin (concentration is 0.5%) press to 1:1(v/v) mixed enzyme (two enzyme) of composition, add and be full of umbilical vein, with mosquito forceps, clamp the umbilical cord the other end; 37 ℃ 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, exposed umbilical vein internal surface, with cell, scrape 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 under 4 ℃, the condition of 500g, centrifugal 5 minutes.
In embodiment 4 is different, picalon forms the comparison of the separation method of cell
1, materials and methods
Umbilical cord is taken from full-term normal delivery or caesarean healthy puerpera, is put in containing 4 ℃ of preservations in dual anti-PBS solution after collection, in 48h, processes.Repeatedly clean, and with phosphoric acid buffer PBS irrigation umbilical vein, until flowing liquid is transparent.With diverse ways, carry out separation respectively.
A. enzyme digestion: clamp umbilical cord one end with mosquito forceps, with liquid-transfering gun, II Collagenase Type (0.2%) added and be full of umbilical vein, clamp the umbilical cord the other end with mosquito forceps; 37 ℃ of shaking tables digest 15 minutes; After having digested, take off mosquito forceps, collect Digestive system in 50ml centrifuge tube, then rinse vein with 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: umbilical cord is cut open along umbilical vein, exposed umbilical vein internal surface, with cell, scrape umbilical vein 5 minutes; With PBS solution, clean, 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 be full of umbilical vein, clamp the umbilical cord the other end with mosquito forceps; 37 ℃ 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, exposed umbilical vein internal surface, with cell, scrape umbilical vein 5 minutes; With PBS, clean, the consumption of PBS solution is 1 times (ml/cm) of umbilical vein length, collects in washings and centrifuge tube.
After three kinds of methods are processed, 4 ℃ respectively of the centrifuge tubes of collecting cell, 500g, centrifugal 5 minutes.Use respectively EGM2-MV substratum resuspended, be inoculated in the 35mm culture dish after I type mouse tail collagen bed board, the amount inoculating cell by a 35mm ware of every 2cm umbilical cord inoculation, is placed in 37 ℃, the CO of volume fraction 5%
2in saturated humidity incubator, cultivate.According to Growth of Cells situation, every 2-3 days full dose is changed liquid once.Growth is when the cell of fast group reaches 90% degree of converging, and the pancreatin with 0.05%/EDTA digestion, counts and respectively organize P0 for the quantity of 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 interior picalon and forms cell.
It is as shown in table 1 that the interior picalon of three kinds of method acquisitions forms cell P0 algebraic quantity:
The cell quantity comparison that table 1 different methods obtains
| ? | 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 following 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 is 31.5 ten thousand for cell, is to adopt separately 3.7 times and 2.3 times of the first two kind method.
The interior picalon that the inventive method obtains forms cell quantity, highly significant follows the example of higher than using separately machinery to scrape the interior picalon formation cell quantity obtaining with enzyme digestion, illustrate that the present invention combines by machinery being scraped follow the example of with enzyme digestion, improved very significantly the separation efficiency that interior picalon forms cell.
In embodiment 5 the present invention, picalon forms the optimization of 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 the two enzymes that form by 1:1 with 0.5% pancreatin; Digestion time is respectively 15 minutes, 30 minutes and 60 minutes.
Growth is when the cell of fast group reaches 90% degree of converging, and the pancreatin with 0.05%/EDTA digestion, counts and respectively organize P0 for the quantity of cell.
2, experimental result
With after different enzymic digestion condition digestion, then scraping collecting cell.After two enzymic digestions, through cultivating, obtained more cell (table 2).
Table 2 adopts the method for collagenase digesting+mechanical scraping, the result that different enzymic digestion conditions is optimized
| 15min30min60min collagenase 11.8 ten thousand 24.0 10,000 3.3 ten thousand pancreatin+collagenases 31.5 ten thousand 24.8 10,000 2.8 ten thousand |
As shown in table 2, while adopting separately the digestion of II Collagenase Type, the cell quantity of acquisition, along with the prolongation of digestion time, first increases afterwards and reduces, and during digestion 30min, the cell quantity of acquisition is maximum, is 24.0 ten thousand; With mixed enzyme when digestion of 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 the cell quantity that adopts separately the digestion of II Collagenase Type to obtain.
Experimental result explanation, adopts the mixed enzyme of pancreatin and II Collagenase Type to digest 15min, and it is maximum that the interior picalon of acquisition forms cell quantity.
The interior picalon of embodiment 6 the inventive method separation forms the evaluation of cell
1, experimental technique
(1) interior picalon forms the cultivation of going down to posterity of cell
Get the P0 of embodiment 3 the inventive method acquisitions for cell, by 10000/cm
2by the density inoculation of going down to posterity.Go down to posterity in culturing process, within every 3 days, full dose is changed liquid, until attached cell while reaching 90% degree of converging, is repeated aforesaid operations, goes down to posterity.
(2) interior picalon forms cell surface marker detection
Enzymic digestion+machinery scrape follow the example of separation and Culture P5 for cell routine digestion after, with CD14, CD73, the VEGFR2 of PE mark, the CD31 of FITC mark, CD90, CD34, CD45 antibody and corresponding homotype contrasting marking cell thereof, carry out flow cytometer detection.
(3) interior picalon forms the experiment of cell low-density lipoprotein (LDL) endocytosis
When enzymic digestion+machinery is scraped the P5 that follows the example of separation and Culture and reached 70% for Growth of Cells to degree of converging, add the LDL of 10 μ g/ml Dil-Ac marks, hatch 4 hours for 37 ℃; Remove substratum, PBS washing 3 times, fixes 30 minutes with 3% paraformaldehyde in room temperature; With PBS, repeatedly clean 3 times, at fluorescence microscopy Microscopic observation.
(4) interior picalon forms cells in vitro vascularization experiment
Matrigel is placed on ice to the thawing of spending the night; Matrigel after melting is added in 24 orifice plates of precooling, hatch 1 hour for 37 ℃; Interior picalon formation of P5 generation cell is digested with pancreatin/EDTA of 0.05%, by the amount in 10000 every holes of cell, be inoculated in ready Matrigel culture plate; Be placed in 37 ℃, in the CO2 saturated humidity incubator of volume fraction 5%, cultivate after 4 hours, 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, the interior picalon in P5 generation forms cell surface expression CD31, VEGFR2 and the CD73 sign of cell; Do not express CD14, CD90, CD45, and CD34 is the expression of diffuse type, the interior picalon that meets bibliographical information forms the feature of the surface marker expression of cell.
As shown in Figure 3, after hatching, the latter is carried out to endocytosis with the LDL after mark, 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 interconnect, form nettedly, shown that cell has very strong extracorporeal blood vessel and forms ability.
Experimental result explanation, the cell that separation of the present invention obtains, has the surface marker that interior picalon forms cell, has LDL endocytosis and very strong extracorporeal blood vessel formation ability, belongs to interior picalon and forms cell.
Experimental result explanation, the inventive method can be from umbilical vein, and effectively separated interior picalon forms cell, and the cell quantity of acquisition is many, and application prospect is good.
Claims (10)
1. in, picalon forms a separation method for cell, it is characterized in that: comprise the steps:
(1) enzymic digestion: get umbilical vein, rinse to the liquid-transparent flowing out with alkalescence or neutral buffered perfusion, pour into the mixed enzyme of collagenase or collagenase and pancreatin, sealing two ends, digest 15～60min, open two ends, collect Digestive system;
(2) mechanical scraping: umbilical vein is cut open, exposed umbilical vein internal surface, with cell scraping umbilical vein internal surface 3～10 minutes, then with alkalescence or the washing of neutral buffered liquid, collect washings;
(3) washings of the Digestive system of step (1) and step (2) is merged, centrifugal, remove supernatant.
2. separation method according to claim 1, is characterized in that: in step (1), 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), described collagenase is II Collagenase Type.
4. separation method according to claim 1, is characterized in that: 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.
5. separation method according to claim 1, is characterized in that: in step (1), the temperature of described digestion is 37 ℃.
6. separation method according to claim 1, is characterized in that: in step (1), the time of described digestion is 15～30min.
7. separation method according to claim 1, is characterized in that: in step (2), the time of described scraping is 5 minutes.
8. separation method according to claim 1, is characterized in that: in step (2), described alkalescence or neutral buffered liquid are PBS damping fluid, DPBS damping fluid or Hanks damping fluid.
9. separation method according to claim 1, is characterized in that: in step (2), the consumption of described damping fluid is 0.5～1.5 times (ml/cm) of umbilical vein length.
10. separation method according to claim 9, is characterized in that: the consumption of described damping fluid is 1 times (ml/cm) of umbilical vein length.
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