CN110904572B

CN110904572B - Double-layer electro-spun fibrous membrane for efficient stem cell amplification and preparation method and application thereof

Info

Publication number: CN110904572B
Application number: CN201911228868.6A
Authority: CN
Inventors: 李燕; 张�浩; 宋婷; 臧宏运; 刘宇洋; 崔镇华
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2019-12-04
Filing date: 2019-12-04
Publication date: 2021-03-23
Anticipated expiration: 2039-12-04
Also published as: CN110904572A

Abstract

The invention discloses a double-layer electrospun fiber membrane for efficient stem cell amplification and a preparation method and application thereof. The preparation process comprises the following steps: s1, mixing NIPAM, MBA and KPS to obtain a mixed solution 1; s2, preparing a gelatin aqueous solution and a sodium dodecyl sulfate aqueous solution, and mixing the gelatin aqueous solution and the sodium dodecyl sulfate aqueous solution with the mixed solution 1 to obtain a mixed solution 2; s3, heating the mixed solution 2 to 60-70 ℃ for reaction, after the reaction is terminated, diluting and dialyzing the reaction solution, and then freeze-drying the dialyzed solution to obtain a spinning precursor; s4, preparing a gelatin spinning solution, preparing a spinning solution from a spinning precursor, and sequentially spinning the two spinning solutions to obtain a double-layer fiber membrane; and S5, carrying out crosslinking reaction, glycine treatment and cleaning on the double-layer fiber membrane to obtain the double-layer electrospun fiber membrane. The double-layer electrospun fiber membrane can provide a 3D culture environment for cells, the cells can be well adhered and propagated, only the temperature needs to be changed during desorption, the desorption process is simple, no damage is caused to the cells, and the activity of the cells is not influenced.

Description

Double-layer electro-spun fibrous membrane for efficient stem cell amplification and preparation method and application thereof

Technical Field

The invention relates to the technical field of stem cell proliferation, in particular to a double-layer electrospun fiber membrane for efficient stem cell amplification and a preparation method and application thereof.

Background

Stem cells have self-renewal and multiple differentiation potentials, can secrete abundant growth factors and cytokines to promote angiogenesis and cell survival in a paracrine mode, and are applied to treating some diseases which are clinically refractory at present, such as bone repair, diabetes, cardiovascular diseases and the like. Therefore, the in vitro culture technology of stem cells plays an important role in the development of biomedicine and the treatment of diseases.

The adhesion of stem cells to the surface of a substrate material is accomplished by the interaction of integrins (receptors) on the surface of the cell membrane with adhesion proteins (ligands) adsorbed to the surface of the material. Cells adhere to a material, and thus undergo a series of actions such as proliferation, migration, differentiation, and the like. However, efficient detachment of cells from the substrate material is also a very important process.

The traditional cell detachment method mainly comprises a trypsinization method, a mechanical scraper method and a fluid shear force mediated cell detachment method. The trypsinization process consists mainly in the selective hydrolysis of extracellular matrix proteins from cell to basal surface and from cell to cell, causing the cells to become spherical under the tension of the cytoskeleton and to detach from the basal surface. While trypsinization enables rapid detachment of cells, pancreatin digestion affects the activity of the cells and subsequent adsorption, proliferation, and differentiation of the cells on other materials. The mechanical scraping method, which involves harvesting cells by cutting off the bonds between fibronectin and the surface of the culture substrate, protects cell membrane proteins to some extent, but causes some damage to cells. Although the fluid shear force mediated cell detachment method can be used for the detachment process of cells, at present, the magnitude of the shear force applied to the cells is difficult to determine, and the detachment efficiency is low, and the fluid shear force mediated cell detachment method is not suitable for most types of cells, especially for some highly sensitive stem cells/functional cells.

The stimuli-responsive polymer is a material commonly used for cell culture and desorption, and can respond to environmental stimuli to enable the molecular conformation and the physicochemical property to generate severe reversible changes after being stimulated by external micro-environment (such as changes of temperature, PH, ionic strength, light, electric field and the like). By changing the surrounding environment of the stimulus response polymer, the hydrophilicity and the hydrophobicity and the solubility of the polymer can be controlled, and the adsorption of biomolecules on the surface of the polymer and the adhesion of cells can be regulated.

The temperature-sensitive polymers commonly used to mediate cell detachment are mainly Poly (N-isopropylacrylamide), Poly (N-isopropyllactamide), PNIPAm, and Poly (N-vinylcaprolactam), Poly (N-vinylcaprolactam), PNIVCL. The PNIPAm has hydrophilic groups (carbonyl and amido groups) and hydrophobic groups (isopropyl groups) in the molecular chain, so that the PNIPAm has the Lowest Critical Solution Temperature (LCST). When the temperature change crosses the LCST, the aqueous polymer solution undergoes a reversible phase change. The temperature-sensitive substrate based on PNIPAm and the derivative thereof can present completely different surface configurations and surface wettabilities according to the change of temperature; the LCST is about 32 ℃ and is close to the physiological environment temperature of a human body, so the LCST is widely applied to biological materials. At 37 ℃, the surface of the PNIPAm temperature-sensitive substrate is weak hydrophobicity, and cells can grow on the surface in an adhesion mode. When the temperature is lower than LCST, PNIPAm is separated and swells, the substrate surface becomes weak hydrophilic, the anchoring effect between extracellular matrix proteins and the substrate surface is weakened, and cells are detached from the substrate surface.

However, most of the temperature-sensitive substrates are 2D planes based on electron beam irradiation or plasma treatment, and the experimental equipment is relatively expensive and difficult to apply to most laboratories. Since almost all cells are surrounded by other cells and extracellular matrix (ECM) in a three-dimensional (3D) manner in vivo, the lack of a 3D cell culture environment also significantly affects the morphology, proliferation and phenotype of the cells, and thus a 2D culture environment sometimes does not provide predictable data for in vivo experiments, and thus, the construction of a 3D culture environment is extremely important for the proliferation, phenotype and related experiments of the cells.

Disclosure of Invention

The invention aims to provide a double-layer electrospun fiber membrane for efficient stem cell amplification, aiming at the defect of the prior art that a substrate material which can provide a 3D culture environment for stem cells and can well adhere, migrate and proliferate is lacked. The double-layer electrospun fiber membrane can provide a simulated in-vivo 3D culture environment for cells in an in-vitro culture process, and when the double-layer electrospun fiber membrane is used as a substrate, the cells can be well adhered and propagated, only the temperature needs to be changed during desorption, the desorption process is simple, no damage is caused to the cells, and the activity of the cells is not influenced.

The invention also aims to provide a preparation method of the double-layer electrospun fiber membrane for high-efficiency stem cell amplification.

Still another object of the present invention is to provide the use of the double-layered electrospun fiber membrane for efficient expansion of stem cells.

The above object of the present invention is achieved by the following scheme:

a double-layer electrospun fiber membrane for efficient stem cell amplification is prepared by the following preparation method:

s1, mixing and dissolving N-isopropylacrylamide (NIPAM), N-Methylenebisacrylamide (MBA) and an initiator in water to obtain a mixed solution 1, and removing oxygen in the mixed solution;

s2, preparing a gelatin aqueous solution and a sodium dodecyl sulfate aqueous solution, adding the two aqueous solutions into the mixed solution 1, uniformly stirring to obtain a mixed solution 2, and removing oxygen in the mixed solution;

s3, heating the mixed solution 2 to 60-70 ℃ for reaction, cooling to stop the reaction after the reaction is finished, then diluting the reaction solution with water, dialyzing, and freeze-drying the dialyzed solution to obtain a spinning precursor;

s4, preparing a gelatin spinning solution, preparing a spinning precursor into a spinning solution, sequentially spinning the two spinning solutions, and obtaining a double-layer electrospun fiber membrane by adopting the same receiver;

and S5, carrying out crosslinking reaction on the double-layer fiber membrane obtained in the step S4 and a glutaraldehyde ethanol solution, cleaning after the reaction is finished, then adding glycine for treatment, and finally cleaning to obtain the double-layer electrospun fiber membrane.

Preferably, in step S1, N-isopropylacrylamide, N-methylenebisacrylamide and the initiator are used in a molar ratio of 100:4: 1.

Preferably, in step S1, the initiator is potassium persulfate (KPS).

Preferably, in the mixed solution 2 of step S2, the mass ratio of gelatin to N-isopropylacrylamide is 1: 1.

Preferably, in step S2, the gelatin aqueous solution and the sodium dodecyl sulfate aqueous solution are prepared at 40 to 60 ℃.

Preferably, in step S3, the heating temperature is 60 ℃; the temperature of the temperature reduction is 0 ℃.

Preferably, in step S4, the gelatin spinning solution has a concentration of 10% and the solvent used is hexafluoroisopropanol.

Preferably, in step S4, the spinning solution prepared from the spinning precursor has a concentration of 10% and the solvent used is hexafluoroisopropanol.

Preferably, in step S4, the spinning parameters are: spinning voltage is 16kv, receiving distance is 20cm, advancing speed is 1mL/h, and a medical 8-gauge needle is used; the receiver is a glass sheet aluminum foil.

Preferably, in step S5, the volume ratio of glutaraldehyde to ethanol in the glutaraldehyde ethanol solution is 1: 50; the temperature of the crosslinking reaction is 4 ℃, and the reaction time is 16-36 h.

Preferably, in step S5, the washing is performed with a phosphoric acid buffer solution; the temperature of the glycine treatment process is 37 ℃, and the treatment time is 1.5 h.

The invention also protects the application of the double-layer electrospun fiber membrane as a 3D substrate for in-vitro cell culture and desorption.

Preferably, the cell comprises a stem cell, a fibroblast or a Hep-G2 cell.

Compared with the prior art, the invention has the following beneficial effects:

according to the double-layer electrospun fiber membrane, the gelatin is added in the process of N-isopropylacrylamide polymerization reaction to prepare the spinning solution, and then the double-layer electrospun fiber membrane is prepared by a spinning technology, so that the defect that the PNIPAm cannot be used for spinning to obtain the fiber membrane is overcome, and the application is simpler and more convenient;

simultaneously, the double-deck electric spin fibrous membrane of preparation can provide the internal 3D culture environment of simulation for the cell in the in vitro culture process, just when double-deck electric spin fibrous membrane was as the basement, the adhesion that the cell can be fine and breed, only need during the desorption change the temperature can, the desorption process is simple, and does not have any damage to the cell, does not influence the activity of cell, especially stem cell's cultivation, adhesion that not only can be fine, appreciation, simultaneously can also be simple, convenient let stem cell carry out the not damaged desorption.

Drawings

FIG. 1 is a graph showing the particle size measurements of the PNI-Gel nanoparticles of example 2 at different temperatures.

FIG. 2 is an SEM image of a fiber membrane prepared by a cross-linking reaction of the Gel monolayer fiber membrane in example 3 under different conditions.

FIG. 3 is an SEM image of a fiber membrane prepared by a cross-linking reaction of the Gel monolayer fiber membrane in example 3 under different conditions.

FIG. 4 shows the results of the cell adhesion and detachment test in example 4.

FIG. 5 shows the results of the proliferation assay after cell adhesion in example 5.

FIG. 6 is a macroscopic cross-sectional view and a topographical view of a two-layer fiber membrane in example 6.

Detailed Description

The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

A double-layer electrospun fiber membrane for efficient stem cell amplification is prepared by the following specific steps:

1. the preparation method of the PNI-Gel nano-particles comprises the following steps:

(1) adding 35mL of deionized water into the round-bottom flask, introducing nitrogen for bubbling, and removing oxygen; adding 35mL of deionized water into a two-neck round-bottom flask, adding 1.13g of NIPAM (N-isopropylacrylamide), 0.0616g of MBA (N, N-methylenebisacrylamide) and 0.027g of KPS (potassium persulfate), introducing nitrogen for 20min, and magnetically stirring for 600r/min in the nitrogen introduction process until the materials are completely dissolved;

(2) 1.13g of Gel (type A) was dissolved in 10mL of deionized water, and 0.0335g of SDS (sodium dodecyl sulfate) was dissolved in 5mL of deionized water, and heated to 40 ℃ or higher to sufficiently dissolve the Gel. Then adding the Gel solution and the SDS solution into the solution 1, introducing nitrogen for 30min, and magnetically stirring for 600r/min until the solution is completely dissolved;

(3) the round-bottom flask is immersed in water at 60 ℃ to ensure that the temperature of the reaction solution is raised to 60 ℃, and the reaction solution is placed into ice water to terminate the reaction after 1 hour of reaction. And adding 50mL of deionized water diluted solution into the reaction solution twice, filling the reaction solution into a dialysis bag with the interception amount of 3500, dialyzing for 7 days, replacing the deionized water every day, and freeze-drying.

2. The preparation method of the PNI-Gel single-layer fiber film comprises the following steps:

(1) the lyophilized PNI-Gel ═ 1:1 was dissolved in HFIP (hexafluoroisopropanol) to a final concentration of 10 wt%. 10 wt% Gel/HFIP was formulated.

(2) Spinning parameters are as follows: spinning with a spinning voltage of 16kv, a receiving distance of 20cm, a propelling speed of 1mL/h and a medical No. 8 needle head, spinning 320uL of 10 wt% PNI-Gel/HFIP and receiving the PNI-Gel/HFIP on an aluminum foil attached with a plurality of glass sheets with the diameter of 14mm to obtain the PNI-Gel single-layer fiber membrane.

3. The preparation method of the PNI-Gel double-layer fiber film comprises the following steps:

(2) Spinning parameters are as follows: spinning voltage is 16kv, receiving distance is 20cm, advancing speed is 1mL/h, and a medical 8-gauge needle is used; the double-layer fiber membrane was obtained by spinning 10 wt% Gel/HFIP on an aluminum foil attached with a plurality of glass sheets 14mm in diameter, and then spinning 10 wt% PNI-Gel/HFIP solution on the same aluminum foil.

(3) Placing a plurality of glass sheets receiving the Gel single-layer fiber membrane into a 12-hole plate, adding 1mL of mixed solution of glutaraldehyde and ethanol (volume ratio is 1:50) into each hole, and then placing the hole plate at 4 ℃ for treatment for 16h, 24h and 36h to fully crosslink the fiber membrane. After crosslinking is completed, PBS is washed for 3 times, 1mL of glycine (7.5g/500mL of PBS) is added for treatment at 37 ℃ for 1.5h, and after the treatment is completed, PBS is used for washing for 3 times to remove the redundant glycine, so that the double-layer electrospun fiber membrane, namely the PNI-Gel double-layer fiber membrane, is obtained.

Example 2

The PNI-Gel nanoparticles prepared according to example 1 were measured for particle size at different temperatures using a particle size meter, and the results are shown in fig. 1.

As can be seen from FIG. 1, the prepared PNI-Gel particles have obvious particle size changes at 20 ℃ and 37 ℃, and when the temperature is reduced from 37 ℃ to 20 ℃, the particle size of the PNI-Gel particles is obviously changed, which indicates that the PNI-Gel nanoparticles have good temperature response performance.

Example 3

1. Influence of glutaraldehyde aqueous solution crosslinking on appearance of gelatin fiber membrane

Preparation of Gel monolayer fiber film:

(1) a circular glass plate with a diameter of 14mm and a thickness of 200um was placed on the aluminum foil for receiving the fiber film.

(2) The spinning solution is gelatin solution with the mass concentration of 10%, and the solvent is HFIP (hexafluoroisopropanol); spinning 320uL Gel single-layer fiber membrane, wherein the spinning voltage is 16kv, the receiving distance is 20cm, and the advancing speed is 1 mL/h.

(3) Placing a plurality of glass sheets receiving the Gel single-layer fiber membrane into a 12-hole plate, adding 1mL of mixed solution of glutaraldehyde and ethanol (volume ratio is 1:50) into each hole, and then placing the hole plate at 4 ℃ for treatment for 16h, 24h and 36h to fully crosslink the fiber membrane. After crosslinking was complete, PBS was washed 3 times, 1mL glycine (7.5g/500mL PBS) was added and treated at 37 ℃ for 1.5h, and after treatment was complete, PBS was washed 3 times to remove excess glycine.

Then respectively putting the glass slides into water bath at 37 ℃, and freeze-drying the glass slides to obtain samples; SEM images of the samples are respectively collected before and after the water bath so as to observe the shape change of the samples. The results are shown in FIG. 2.

2. Influence of glutaraldehyde steam crosslinking on appearance of gelatin fiber membrane

The Gel monolayer fiber membrane is prepared according to the preparation method, then a plurality of glass sheets receiving the Gel monolayer fiber membrane are put into a plastic transparent bowl with a seal, the plastic transparent bowl is placed in a culture dish containing 10mL of 25% glutaraldehyde aqueous solution, and silica Gel is put into the bowl to remove the water in the closed space. Crosslinking 1day, 2day and 3day at room temperature, then respectively placing the crosslinked fibers into a water bath at 37 ℃, and freeze-drying the crosslinked fibers after the fibers are separated from the slide to obtain a sample; SEM images of the samples are respectively collected before and after the water bath so as to observe the shape change of the samples. The results are shown in FIG. 3.

As can be seen from FIG. 2, the Gel monolayer fiber membrane crosslinked by glutaraldehyde aqueous solution for 24h can still maintain good fiber morphology after being subjected to water bath; as can be seen from FIG. 3, the morphology of the Gel monolayer fiber membrane crosslinked by the glutaraldehyde steam method after water bath is poor, and comparison shows that the Gel monolayer fiber membrane crosslinked by the glutaraldehyde aqueous solution for 24 hours can be used in subsequent cell experiments.

Example 4

(1) The PNI-Gel monolayer fiber membrane was prepared according to the method of example 1, and then the glass sheet receiving the PNI-Gel monolayer fiber membrane was put into a 12-well plate, 1mL of a mixed solution of glutaraldehyde and ethanol (volume ratio 1:50) was added to each well, and then the well plate was put at 4 ℃ and treated for 1day to fully crosslink the fiber membrane.

(2) After crosslinking was complete, PBS was washed 3 times, 1mL glycine (7.5g/500mL PBS) was added and treated at 37 ℃ for 1.5h, and after treatment was complete, PBS was washed 3 times to remove excess glycine.

(3) And (3) flatly paving the glass sheet at the bottom of the 24-hole plate, and lightly compacting to be tightly fit with the bottom. SD rat adipose stem cells were labeled with cell tracker red at 2 x 10⁴The density of the pores is inoculated on a fiber membrane and the fiber membrane is placed at 37 ℃ and 5 percent CO₂The culture was carried out in an incubator for 24 hours and photographed. The well plate was then incubated at 4 ℃ for 1h and the cell density on the fiber membrane was observed.

As shown in FIG. 4, it is understood from FIG. 4 that the cells grew well on the fibrous membrane at 37 ℃ and the adhered cells were detached from the fibrous membrane at 20 ℃.

Example 5

(1) The PNI-Gel single layer fiber membrane was prepared according to the method of example 1, the Gel single layer fiber membrane was prepared according to the method of example 3, then the glass sheet receiving the PNI-Gel single layer fiber membrane or the Gel single layer fiber membrane was placed in a 12-well plate, respectively, 1mL of mixed solution of glutaraldehyde and ethanol (volume ratio 1:50) was added to each well, and then the well plate was placed at 4 ℃ and treated for 1day to fully crosslink the fiber membranes.

(2) After crosslinking was completed, PBS was washed 3 times, 1mL of glycine (7.5g/500mL of PBS) was added and treated at 37 ℃ for 1h30min, and after completion of treatment, PBS was washed 3 times to remove excess glycine.

(3) And (3) flatly paving the glass sheet at the bottom of the 24-hole plate, and lightly compacting to be tightly fit with the bottom. SD rat adipose-derived stem cells with 2 x 10⁵The density of the/well is inoculated on a fiber membrane and placed at 37 ℃ with 5% CO₂Culturing in an incubator. Cell proliferation was measured for day5 and day 7.

As shown in FIG. 5, it can be seen from FIG. 5 that adipose-derived stem cells proliferated well on the PNI-Gel monolayer fibrous membrane and were superior to the pure Gel monolayer fibrous membrane.

Example 6

(1) A PNI-Gel bilayer fiber membrane was prepared according to the method of example 1, and a glass plate receiving the PNI-Gel bilayer fiber membrane was placed in a 12-well plate, and 1mL of glutaraldehyde (10mL/500mL of ethanol) was added per well, and then the plate was placed at 4 ℃ and treated for 1day to sufficiently crosslink the fiber membrane.

(3) And then adding 1mL of PBS to soak in a water bath kettle at 37 ℃ for 1day to make the fibrous membrane fall off, wherein the OCT represents the deformation performance of the fibrous membrane, and the CLSM represents the macroscopic morphology of the fibrous membrane.

As shown in fig. 6, it is understood from fig. 6 that the double-layered fiber film is curled at 37 ℃ and deformed to form a three-dimensional structure.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A double-layer electrospun fiber membrane for efficient stem cell amplification is characterized by comprising the following preparation method:

s1, dissolving N-isopropyl acrylamide, N-methylene bisacrylamide and an initiator in water to obtain a mixed solution 1, and removing oxygen in the mixed solution;

s4, preparing a gelatin spinning solution, preparing a spinning precursor into a spinning solution, sequentially spinning the two spinning solutions, and obtaining a double-layer fiber membrane by adopting the same receiver;

2. The double-layered electrospun fiber membrane for efficient stem cell expansion according to claim 1, wherein in step S1, the ratio of the amounts of N-isopropylacrylamide, N-methylenebisacrylamide and the initiator is 100:4:1 by mole.

3. The double-layered electrospun fiber membrane for efficient expansion of stem cells according to claim 1, wherein the mass ratio of gelatin to N-isopropylacrylamide in the mixed solution 2 of step S2 is 1: 1.

4. The double-layered electrospun fiber membrane for efficient expansion of stem cells according to claim 1, wherein in step S1, the initiator is potassium persulfate; in step S2, the gelatin aqueous solution and the sodium dodecyl sulfate aqueous solution are prepared at 40-60 ℃.

5. The double-layered electrospun fiber membrane for efficient expansion of stem cells according to claim 1, wherein the temperature of heating is 60 ℃ in step S3; the temperature of the temperature reduction is 0 ℃.

6. The double-layered electrospun fiber membrane for efficient stem cell expansion according to claim 1, wherein in step S4, the mass concentration of gelatin in the gelatin spinning solution is 10%, and the adopted solvent is hexafluoroisopropanol; the concentration of solute in the spinning solution prepared from the spinning precursor is 10%, and the adopted solvent is hexafluoroisopropanol.

7. The double-layered electrospun fiber membrane for efficient stem cell expansion according to claim 1, wherein in step S4, the spinning parameters are: spinning voltage is 16kV, receiving distance is 20cm, propelling speed is 1mL/h, and a medical 8-gauge needle head is adopted; the receiver is a glass sheet aluminum foil;

8. The double-layered electrospun fiber membrane for efficient stem cell expansion according to claim 1, wherein in step S5, the volume ratio of glutaraldehyde to ethanol in the glutaraldehyde ethanol solution is 1: 50; the temperature of the crosslinking reaction is 4 ℃, and the reaction time is 16-36 h.

9. Use of the double-layered electrospun fiber membrane of any one of claims 1 to 8 as a 3D substrate for in vitro culture and detachment of cells.

10. The use of claim 9, wherein the cell is a stem cell, a fibroblast cell, or Hep-G2.