CN108359635B - One kind being used for enriching and purifying c-kit+Composite coating of heart cell and preparation method thereof - Google Patents
One kind being used for enriching and purifying c-kit+Composite coating of heart cell and preparation method thereof Download PDFInfo
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0657—Cardiomyocytes; Heart cells
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- C12N2535/00—Supports or coatings for cell culture characterised by topography
Abstract
The invention discloses one kind to be used for enriching and purifying c-kit+Composite coating of heart cell and preparation method thereof, belong to technical field of biological material, composite coating of the present invention includes hydrophilic polymer A, hydrophilic polymer B and water, hydrophilic polymer A is the polyethers that alginates or polyoxypropylene and polyoxyethylene are constituted, and hydrophilic polymer B is gelatin, fibronectin or poly-D-lysine.Preparation method of composite coating of the present invention is simple, and composite coating can simplify c-kit+The separation method of heart cell, and the c-kit of enriching and purifying+Heart cell aggregation physical efficiency treats ischemic injuries cardiomyopathies well.
Description
Technical field
The invention belongs to technical field of biological material, more particularly to one kind to be used for enriching and purifying c-kit+The compound painting of heart cell
Layer and preparation method thereof.
Background technique
Myocardial damage caused by ischemic cardiomyopathy is one of highest disease of the death rate in current heart disease, and cell
Regenerative therapy is considered as one of most potential treatment method.The Proliferation, Differentiation of Cardiac Stem Cells is induced, or stimulates its point
Growth factor is secreted to be considered to have protection and repair damaged myocardium tissue.Studies have shown that c-kit in heart+Cell has protection
With the potential for repairing damaged myocardium.Currently, c-kit+The Isolation and purification method of heart cell is mainly immunomagnetic beads screening method,
This method is to be combined using cell surface antigen c-kit with corresponding screening magnetic bead and screened to cell.Due to sieve
The cell elected is dispersity and the magnetic bead for carrying sorting, and the existing defects in myocardial damage treatment: dosage is big, glutinous
Attached ability is weak, activity is low, considerably reduces the therapeutic effect of stem cell regenerating therapy.
Therefore, it is necessary to simplify c-kit+The separation method of heart cell, and make the c-kit of enriching and purifying+Heart cell energy
Myocardial damage is treated well.
Summary of the invention
Present invention aims to overcome that the shortcomings of the prior art, and provide a kind of for enriching and purifying c-kit+Heart
Composite coating of cell and preparation method thereof, the preparation method of composite coating is simple, and composite coating can simplify c-kit+Heart is thin
The separation method of born of the same parents, and the c-kit of enriching and purifying+Heart cell can treat damaged myocardium well.
To achieve the above object, the technical scheme adopted by the invention is as follows: one kind be used for enriching and purifying c-kit+Heart cell
Composite coating, it includes having hydrophilic polymer A, hydrophilic polymer B and water, the hydrophilic polymer A is alginates or polyoxy
The polyethers that propylene and polyoxyethylene are constituted, the hydrophilic polymer B are gelatin, fibronectin or poly-D-lysine.
New coating compound of the present invention (contains high hydrophilic group by changing constituent used in composite coating
The hydrophilic polymer A and hydrophilic polymer B containing medium hydrophilic radical of group) ratio, do not needing by any antibody situation
Under, it can be simply and efficiently by c-kit+Heart cell is poly- to be separated, and is c-kit+Heart cell collective is current separation
Not available for purification process: existing method is only to isolate the c-kit individually dispersed+Heart cell, these c-kit+The heart
The Adhering capacity of dirty cell is weak, activity is low.
As an improvement of the above technical solution, the polyethers is at least one in poloxamer F68 and poloxamer F127
Kind.
As a further improvement of the above technical scheme, the hydrophilic polymer A is poloxamer F68, described hydrophilic poly-
Conjunction object B is gelatin.
In addition, the present invention also provides the preparation methods of the composite coating comprising following steps:
S1 it) dissolves: hydrophilic polymer A is dissolved in the water, obtain hydrophilic polymer A lysate after mixing evenly；It will be close
Aqueous polymer B is dissolved in the water, and obtains hydrophilic polymer B lysate after mixing evenly；
S2 it) mixes: hydrophilic polymer A lysate being mixed with hydrophilic polymer B lysate, is stirred to get composite coating
Lysate；
S3 it) sterilizes and is incubated for: the composite coating lysate is filtered, filtered composite coating lysate is added
To the surface of cell culture container, it is placed in incubator and is incubated for；
S4) dry: the composite coating lysate after incubation being placed in gnotobasis, inhales and abandons remaining water in culture vessel
Solution, and be dried, it can be used to enriching and purifying c-kit+Heart cell.
As an improvement of the above technical solution, during the hydrophilic polymer A is dissolved in water, hydrophilic polymer A with
The mass ratio of water is 0.1%~10%.
As an improvement of the above technical solution, during the hydrophilic polymer B is dissolved in water, hydrophilic polymer B with
The mass ratio of water is 0.1%~10%.
As an improvement of the above technical solution, in step s2) mixing, the hydrophilic polymer A lysate and the parent
The mass ratio of aqueous polymer B lysate is 0.1%~99.9%.
As an improvement of the above technical solution, the step s1) dissolution temperature be 60 DEG C；The step s3) be incubated for
Temperature is 37 DEG C, time 18h；The step s4) the dry time is at least 2h.
The beneficial effects of the present invention are: the present invention provides a kind of for enriching and purifying c-kit+The compound painting of heart cell
Layer and preparation method thereof, new coating compound of the present invention, by changing constituent used in composite coating (containing height
The hydrophilic polymer A of hydrophilic radical and hydrophilic polymer B containing medium hydrophilic radical) ratio, do not needing by any anti-
It, can be simply and efficiently by c-kit in the case of body+Heart cell is separated, the c-kit of enriching and purifying+Heart cell aggregation physical efficiency
Damaged myocardium is treated well；In addition, composite coating manufacturing process of the present invention is simple, required time is short, low cost, is easy to control
System and implementation, and product yield is high.
Detailed description of the invention
Figure 1A shows heart cell suspension cell state when rigid addition is coated with the culture plate surface of composite coating；Figure
1B shows that heart cell cultivates rear cell state for 24 hours in the culture plate surface for being coated with composite coating；
Fig. 2A shows the expression using c-kit in the cell aggregation (Agg) formed after composite coating culture of isolated；
Fig. 2 B, which is shown, is attached on c-kit in the cell (Att) on composite coating surface using the dispersion formed after composite coating culture of isolated
Expression；Fig. 2 C counts the heart cell ratio that c-kit is expressed in all cell aggregations and all single cell dispersions；
Fig. 3 be show immunofluorescence dyeing to separation formed cell aggregation and single cell dispersion group in cell into
Row dyeing；Wherein, from top to bottom, it is followed successively by bright-field figure, marks the figure of c-kit, is marked TnI (labelled protein of myocyte)
Figure, mark c-kit and TnI fusion figure；In addition, in right part of flg white circle mark be cell aggregation rather than it is single
Cell dispersion；
State of the heart cell of the primary culture (P1) of Fig. 4 A display passage on composite coating；Fig. 4 B display passage is secondary
Cultivate state of the heart cell of (P2) on composite coating；The heart cell of Fig. 4 C display five cultures (P5) of passage is compound
State on coating；After Fig. 4 D shows that heart cell passes on primary, secondary and five cultures, through obtained by composite coating enriching and purifying
C-kit in cell aggregation+The ratio (%) of heart cell；
After Fig. 5 shows heart cell ischemic preconditioning, the cell LDH heart unit emission under different disposal；Wherein, blank pair
According to group (Con): heart cell is in the control culture 120h without any processing；Negative control group (IS): heart cell is damaged in ischemic
120h is cultivated without any post-processing after wound；Test group (Agg): heart cell is added immediately the c- of enriching and purifying after ischemic injuries
Kit+ heart cell aggregation body, co-incubation 120h；Note: asterisk indicates that there are significant differences: p with blank control group in figure
< 0.05；
Fig. 6 display heart cell is after ischemic injuries, the c-kit of addition+The shape of heart cell aggregation body and heart cell
State；
After Fig. 7 shows heart cell ischemic preconditioning, the cell LDH heart unit emission under different disposal；Wherein, blank pair
According to group (Con): heart cell is in the control culture 120h without any processing；Negative control group 1 (IS): heart cell is damaged in ischemic
120h is cultivated without any post-processing after wound；Negative control group 2 (Non-treat): nothing is added after ischemic injuries and appoints for heart cell
The culture solution culture 120h of what cell factor；Test group (AggCM): heart cell is added immediately the c-kit+ heart after ischemic injuries
The synthesis culture solution of dirty cell aggregation secretion, co-incubation 120h；Note: asterisk indicates to exist with blank control group significant in figure
Sex differernce: p < 0.05.
Specific embodiment
Purposes, technical schemes and advantages in order to better illustrate the present invention, below in conjunction with specific embodiments and the drawings pair
The present invention is described further.
Embodiment 1
The present embodiment provides one kind to be used for enriching and purifying c-kit+The preparation method of the composite coating of heart cell comprising
Following steps:
S1 it) dissolves: poloxamer F68 is dissolved in the water, obtain hydrophilic polymer A lysate after mixing evenly；It will be bright
Glue is dissolved in the water, and obtains hydrophilic polymer B lysate after mixing evenly；Temperature when dissolution is 60 DEG C；Poloxamer F68 is molten
During Xie Yushui, the mass ratio of poloxamer F68 and water is 0.1%；Gelatin is during water, the quality of gelatin and water
Than being 6%；
S2 it) mixes: hydrophilic polymer A lysate being mixed with hydrophilic polymer B lysate, is stirred to get composite coating
Lysate；The mass ratio of the hydrophilic polymer A lysate and the hydrophilic polymer B lysate is 0.1%；
S3 it) sterilizes and is incubated for: aperture being used to be filtered for 7 μm of filters to composite coating lysate, it is filtered multiple
The surface that coating lysate is added to cell culture container is closed, is placed in incubator and is incubated for；Temperature when incubation is 37
DEG C, time 18h；
S4) dry: the composite coating lysate after incubation being placed in superclean bench together with cell culture container, inhales and abandons
Remaining aqueous solution in cell culture container, and be dried, it can be used to enriching and purifying c-kit+Cardiac stem cells；When dry
Time be at least 2h.
In addition, the present embodiment also provides a kind of composite coating prepared using the preparation method.
Embodiment 2
The present embodiment provides one kind to be used for enriching and purifying c-kit+The preparation method of the composite coating of heart cell, with implementation
Example 1 is similar, and difference is:
S1) dissolve: hydrophilic polymer A is Luo Shamu F127, and poloxamer F127 is dissolved in during water, poloxamer
The mass ratio of F127 and water is 5%；Hydrophilic polymer B is fibronectin, and fibronectin is dissolved in during water, fibronectin with
The mass ratio of water is 0.1%；
S2) mix: the mass ratio of the hydrophilic polymer A lysate and the hydrophilic polymer B lysate is 20%；
In addition, the present embodiment also provides a kind of composite coating prepared using the preparation method.
Embodiment 3
The present embodiment provides one kind to be used for enriching and purifying c-kit+The preparation method of the composite coating of heart cell, with implementation
Example 1 is similar, and difference is:
S1) dissolve: hydrophilic polymer A is alginates, and alginates are dissolved in during water, and the mass ratio of alginates and water is
7%；Hydrophilic polymer B is poly-D-lysine, and poly-D-lysine is dissolved in during water, and the mass ratio of poly-D-lysine and water is
10%；
S2) mix: the mass ratio of the hydrophilic polymer A lysate and the hydrophilic polymer B lysate is 60%；
In addition, the present embodiment also provides a kind of composite coating prepared using the preparation method.
Embodiment 4
The present embodiment provides one kind to be used for enriching and purifying c-kit+The preparation method of the composite coating of heart cell, with implementation
Example 1 is similar, and difference is:
S1) dissolve: hydrophilic polymer A be poloxamer F68 and poloxamer F127 mixture, poloxamer F68 and
Poloxamer F127 is dissolved in during water, and the mass ratio of poloxamer F68, the mixture of poloxamer F127 and water are
10%；Hydrophilic polymer B is the mixture of poly-D-lysine and gelatin, and poly-D-lysine and Gelatin are more during water
The mass ratio of polylysine, the mixture of gelatin and water is 5%；
S2) mix: the mass ratio of the hydrophilic polymer A lysate and the hydrophilic polymer B lysate is 99.9%；
In addition, the present embodiment also provides a kind of composite coating prepared using the preparation method.
Embodiment 5
The present embodiment separates heart cell using composite coating described in Examples 1 to 4, enriching and purifying c-kit+Heart cell comprising following steps:
1) heart cell is isolated from the mouse heart in 1 day age using enzymolysis, digestion method, using centrifuge with
The revolving speed of 1200rpm separates cell, and with heart cell culture fluid (IMDM, 20% fetal calf serum, 1% Pen .- Strep,
1%L- glutamic acid) it is resuspended；
2) mixed heart cell is planted in coated 96 orifice plate of composite coating with the density in 5000/hole, 5%
CO2, cultivate for 24 hours in 37 DEG C of incubators, utilize the state of optical microphotograph sem observation cell (as shown in Fig. 1)；
3) using the syringe needle of 0.1mm under the microscope by c-kit+Heart cell aggregation body is separated from composite coating surface,
And individual cells are separated into using enzymatic isolation method；Other single cell dispersions for being individually dispersed in composite coating surface utilize enzymatic isolation method
It is separated from coating surface；Two groups of cells are utilized respectively flow cytometer, are identification antigen to cell count, system with c-kit
Count c-kit in two groups of cells+The quantity (as shown in Figure 2) of heart cell；
4) immunofluorescence analysis is utilized, is identification with c-kit and TroponinI (muscle cell surface antigen, TnI)
Label identifies cell aggregation and single cell dispersion (as shown in Figure 3)；
5) heart cell is isolated from the mouse heart in 1 day age, secondary culture 5 times；Respectively by different passage numbers
Heart cell is planted on 96 orifice plates for being coated with composite coating, and step 2)~4 are repeated), c-kit is counted later+Heart cell
Content (as shown in Figure 4).
As shown in Figure 1, heart cell after composite coating is incubated for, is gathered into c-kit+Heart cell collective, c-kit+Heart
There is also the adherent cells of some dispersions around cell collective.As shown in Fig. 2, separating the c-kit to be formed through composite coating+Heart
In cell aggregation, there are about 87.01% cells to express c-kit；It is separated through composite coating in the single cell dispersion to be formed, there are about
25.04% cell expresses c-kit；It can be seen that c-kit+Most cells express c-kit in heart cell aggregation body.As shown in figure 3,
The a large amount of positive expression c-kit of cell aggregation, and the rare expression c-kit of single cell dispersion.As shown in figure 4, as heart is thin
The increase of born of the same parents' secondary culture number, through the c-kit in cell aggregation obtained by composite coating enriching and purifying+Heart cell ratio by
It is decrescence few, and phenotype changes.
Therefore, composite layer of the present invention can efficiently separate c-kit+Heart cell aggregation body and single cell dispersion, c-
kit+Contain c-kit in heart cell aggregation body+Heart cell；By adjusting the ratio and heart cell of component in composite coating
The number of secondary culture can choose c-kit in cell aggregation+The ratio and phenotype of heart cell.
Embodiment 6
The present embodiment provides a kind of c-kit+The preparation method of the synthesis culture solution of heart cell aggregation body, the c-kit+
Heart cell aggregation body c-kit in embodiment 5+Heart cell aggregation body, synthesize culture solution preparation method include with
Lower step:
1) by separated c-kit+Heart cell aggregation body is placed in the culture plate without any processing, and serum-free is added
Heart cell culture solution (IMDM, 1% Pen .- Strep), collects culture solution afterwards for 24 hours；
2) after the abundant broth filtrate of filter for being 7 μm using aperture, that is, synthesis culture solution is prepared, is placed in -80
It DEG C saves backup.
Embodiment 7
c-kit+The protection of heart cell aggregation body and the heart cell for repairing ischemic injuries
1) 1~2 age in days SD suckling mouse of originally culture aseptically takes out heart and is cleaned, shredded with PBS, moves into later
Containing 0.1% pancreatin, 0.1% Collagenase, 0.1% neutral proteinase (Dispase) mixed enzyme solution in digest 40min；
2) indigested tissue is removed with the filter that aperture is 40 μm, 1300rpm is centrifuged 3min, removes supernatant, uses
Heart cell culture solution (IMDM, 20% fetal calf serum, 1% Pen .- Strep, 1%L- glutamic acid) is resuspended, thin with 5000
Born of the same parents/hole density is moved into 96 orifice plates and is cultivated for 24 hours；
3) PBS flushing 2 times is added after removing cell culture fluid, lacks blood treatment 25min, above method preparation is added later
C-kit+Cell aggregation is transplanted with the density in 20/hole, and heart cell culture solution is added；
4) 100 μ l culture solutions are often removed for 24 hours after oneself is added to mix with LDH detection reagent, measure absorption value in 480nm；
5) it this time tests, sets up test group (Agg), blank control group (Con) and negative control group (IS).
As shown in figure 5, with the extension for the treatment of time, the c-kit that is added in test group+Heart cell aggregation body can subtract
The burst size of few LDH heart unit (lactic dehydrogenase), protection and the heart cell for repairing damage；When treatment time is 96h, test
Group is not significant with the difference of blank control group.
Embodiment 8
To c-kit in the test group of embodiment 7+Heart cell aggregation body and heart cell are observed, as a result such as Fig. 6 institute
Show, c-kit+Heart cell aggregation body is gradually blended on impaired heart cell.
Integrated embodiment 7 and embodiment 8, it is seen that the c-kit of enriching and purifying of the present invention+Heart cell aggregation physical efficiency protection and
Repair the heart cell of ischemic injuries.
Embodiment 9
c-kit+The synthesis culture solution protection of heart cell aggregation body secretion and the heart cell for repairing ischemic injuries
1) 1~2 age in days SD suckling mouse of originally culture aseptically takes out heart and is cleaned with PBS, shredded, immigration contains
0.1% pancreatin, 0.1% Collagenase, 0.1% neutral proteinase (Dispase) mixed enzyme solution in digest 40min；
2) indigested tissue is removed with the filter that aperture is 40 μm, 1300rpm is centrifuged 3min, removes supernatant, uses
Heart cell culture solution (IMDM, 20% fetal calf serum, 1% Pen .- Strep, 1%L- glutamic acid) is resuspended, thin with 5000
Born of the same parents/hole density is moved into 96 orifice plates and is cultivated for 24 hours；
3) PBS flushing 2 times is added after removing culture solution, after lacking blood treatment 25min, the synthesis of above method preparation is added
Culture solution is transplanted with the density in 100 holes μ l/, and heart cell culture solution is added；
4) 100 μ l culture solutions are often removed for 24 hours after oneself is added to mix with LDH detection reagent, are obtained under the absorption light of 480nm
Absorption value；
5) it this time tests, it is right to set up test group (AggCM), blank control group (Con), negative control group 1 (IS) and feminine gender
According to 2 (Non-treat) of group.
As shown in fig. 7, with the extension for the treatment of time, the c-kit that is added in test group+The secretion of heart cell aggregation body
Synthesis culture solution can reduce the burst size of LDH heart unit (lactic dehydrogenase), the damage after protection and reparation heart cell ischemic；
When treatment time is 96h, the difference of test group and blank control group is not significant.
Finally, it should be noted that above embodiments protect the present invention to illustrate technical solution of the present invention
The limitation of range, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should be managed
Solution, can modify to technical solution of the present invention or replace on an equal basis, without departing from technical solution of the present invention essence and
Range.
Claims (4)
1. composite coating is being used for enriching and purifying c-kit+It is applied in heart cell, which is characterized in that the composite coating includes
Hydrophilic polymer A, hydrophilic polymer B and water, the hydrophilic polymer A are that alginates or polyoxypropylene and polyoxyethylene are constituted
Polyethers, the hydrophilic polymer B are gelatin, fibronectin or poly-D-lysine；
The composite coating is to be prepared by the following method:
S1 it) dissolves: hydrophilic polymer A is dissolved in the water, obtain hydrophilic polymer A lysate, hydrophilic polymeric after mixing evenly
The mass ratio of object A and water is 0.1%~10%；Hydrophilic polymer B is dissolved in the water, obtains hydrophilic polymer after mixing evenly
The mass ratio of B lysate, hydrophilic polymer B and water is 0.1%~10%；
S2 it) mixes: hydrophilic polymer A lysate being mixed with hydrophilic polymer B lysate, is stirred to get composite coating dissolution
The mass ratio of liquid, the hydrophilic polymer A lysate and the hydrophilic polymer B lysate is 0.1%~99.9%；
S3 it) sterilizes and is incubated for: the composite coating lysate being filtered, filtered composite coating lysate is added to carefully
The surface of born of the same parents' culture vessel, is placed in incubator and is incubated for；
S4) dry: the composite coating lysate after incubation being placed in gnotobasis, is inhaled remaining water-soluble in abandoning culture vessel
Liquid, and be dried, it can be used to enriching and purifying c-kit+Heart cell.
2. application as described in claim 1, which is characterized in that the polyethers is at least poloxamer F68 and poloxamer
One of F127.
3. application as claimed in claim 1 or 2, which is characterized in that the hydrophilic polymer A is poloxamer F68, the parent
Aqueous polymer B is gelatin.
4. application as described in claim 1, which is characterized in that the step s1) temperature of dissolution is 60 DEG C；The step
S3) temperature being incubated for is 37 DEG C, time 18h；The step s4) the dry time is at least 2h.
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