CN111569157A - Preparation method of biological membrane for constructing tissue-engineered retinal pigment epithelium - Google Patents
Preparation method of biological membrane for constructing tissue-engineered retinal pigment epithelium Download PDFInfo
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Abstract
The invention relates to the field of biology, and particularly discloses a preparation method of a biological membrane for constructing tissue-engineered retinal pigment epithelium, which comprises the following steps: s1: uniformly dissolving polycaprolactone, gelatin and graphene oxide in a polar solvent to obtain a mixed solution, wherein the mass percent concentrations of the polycaprolactone, the gelatin and the graphene oxide in the mixed solution are 69.6%, 29.8% and 0.6% respectively; s2: and (5) placing the mixed solution obtained in the step (S1) in a micro injection pump connected with a high-voltage generator for electrostatic spinning to obtain the biological membrane. The nanofiber composite membrane which not only supports the growth and functions of ESC-RPE cells, but also has biocompatibility and operation operability can be prepared by the method.
Description
Technical Field
The invention relates to the field of biology, in particular to a preparation method of a biological membrane for constructing tissue-engineered retinal pigment epithelium.
Background
The Retinal Pigment Epithelium (RPE) is a single layer of cells located between the retina and the choroid, has the functions of secreting trophic factors, phagocytosing photoreceptor cell outer segments, participating in the visual cycle metabolism and the like, and has very important function for maintaining the normal physiological function of the retina. RPE dysfunction leads to photoreceptor apoptosis, causing retinal degenerative diseases such as age-related macular degeneration (AMD) and Retinitis Pigmentosa (RP). The disease trend of China is rising year by year, but effective treatment measures are not available yet.
Tissue engineering RPE transplantation is a most promising method for treating retinal degenerative diseases, wherein RPE cells are cultured on a biological membrane to form a polar and functional single-layer RPE cell sheet, and then the RPE cell sheet and the biological membrane are transplanted to the subretinal space. Two major elements required for tissue engineering RPE are seed cells and biofilms. In the aspect of seed cells, the RPE cells (ESC-RPE) differentiated from the Embryonic Stem Cells (ESC) have the similar shape and function as in vivo RPE cells, have no risk of genetic variation and high differentiation efficiency, can realize standardized and industrialized production, and are ideal seed cells.
However, the biomembrane prepared by the prior art cannot give consideration to biocompatibility and operation operability on the premise of supporting the growth and the function of RPE cells, thereby limiting the further application of tissue engineering RPE transplantation.
In order to solve the above problems, there is a need for a method for preparing a biofilm for constructing tissue-engineered retinal pigment epithelium, which can prepare a nanofiber composite membrane that supports the growth and function of ESC-RPE cells and can also achieve biocompatibility and surgical operability.
Disclosure of Invention
In view of the above, the present invention aims to provide a method for preparing a biofilm for constructing tissue-engineered retinal pigment epithelium, which can be used for preparing a nanofiber composite membrane that not only supports the growth and functions of ESC-RPE cells, but also has biocompatibility and surgical operability.
In order to achieve the above object, the present invention provides a method for preparing a biofilm for constructing tissue-engineered retinal pigment epithelium, comprising the steps of:
s1: uniformly dissolving polycaprolactone, gelatin and graphene oxide in a polar solvent to obtain a mixed solution, wherein the mass percent concentrations of the polycaprolactone, the gelatin and the graphene oxide in the mixed solution are 69.6%, 29.8% and 0.6% respectively;
s2: and (5) placing the mixed solution obtained in the step (S1) in a micro injection pump connected with a high-voltage generator for electrostatic spinning to obtain the biological membrane.
As a further improvement to the above technical solution, in step S1, the polar solvent is hexafluoroisopropanol.
As a further improvement to the above technical solution, in step S1, the graphene oxide is in a powder form and is prepared by the following steps:
s11: taking 10g of 32-mesh high-purity graphite, and adding the graphite into 500mL of concentrated sulfuric acid at 0 ℃;
s12: then 60g KMnO was added4Heating to 35 deg.C, and stirring;
s13: diluting with water, adding H2O2Adding HCl to the solution to centrifugally wash the solution until the solution is yellow, and dialyzing the solution until the pH value is 6.0;
s14: centrifuging for 10min, taking supernatant, and freeze-drying to obtain graphene oxide powder.
As a further improvement to the above technical solution, in step S2, the electrostatic spinning parameters are: the total concentration is 8-10%, the voltage is 10-15kV, the distance from a spray head to a receiver is 10-20cm, and the electrospinning flow rate is 1-3 mL/h.
Compared with the prior art, the invention has the following beneficial technical effects:
the preparation method of the biomembrane for constructing the tissue engineering retinal pigment epithelium provided by the invention uses polycaprolactone, gelatin and graphene oxide as raw materials through an electrostatic spinning technology, the prepared biomembrane supports the growth and the function of ESC-RPE cells, can be used for constructing the tissue engineering RPE graft, and the graft can be successfully transplanted to the retina lower cavity of an experimental animal, has good biocompatibility, and is expected to provide a new strategy for treating the retinal degeneration disease.
Drawings
FIG. 1 is a scanning electron micrograph of a biofilm prepared according to a first embodiment of the present invention;
FIG. 2 is a statistical graph of the diameter of nanofibers of a biofilm prepared according to one embodiment of the present invention;
FIG. 3 is a graph showing the results of detecting the expression levels of RPE65 and ZO-1 in example two of the present invention;
FIG. 4 is a transmission electron micrograph of ESC-RPE cells in a second example of the present invention;
FIG. 5 is a cluster heatmap of the differential genes of tissue engineered RPE and control in accordance with a second embodiment of the present invention;
FIG. 6 is a heat map of the correlation of gene expression of tissue engineered RPE with a control in accordance with a second embodiment of the present invention;
FIG. 7 is a schematic diagram showing a structure of a retina of a rabbit eye after transplantation in a second embodiment of the present invention;
FIG. 8 is a graph showing a comparison of retinal function between a rabbit eye after transplantation and a normal rabbit eye in a second embodiment of the present invention;
FIG. 9 is toluidine blue staining of a portion of a retina section from a rabbit eye graft according to a second embodiment of the present invention.
Detailed Description
For better understanding of the technical solutions and advantages thereof, the present invention will be described in detail with reference to the accompanying drawings and specific embodiments.
Example one
The present embodiment provides a method for preparing a biofilm for constructing tissue-engineered retinal pigment epithelium, including the following steps S1 and S2:
s1: uniformly dissolving Polycaprolactone (PCL), Gelatin (Gelatin) and Graphene Oxide (GO) in a polar solvent to obtain a mixed solution, wherein the mass percentage concentrations of the polycaprolactone, the Gelatin and the graphene oxide in the mixed solution are 69.6%, 29.8% and 0.6% respectively.
Among them, the polar solvent is preferably Hexafluoroisopropanol (HFIP).
The graphene oxide is in a powder form and can be prepared by the following steps:
s11: taking 10g of 32-mesh high-purity graphite (purchased from Qingdao Tianyuan graphite Co., Ltd.), and adding the graphite into 500mL of concentrated sulfuric acid at 0 ℃;
s12: then 60g of KMnO4 is added, the temperature is raised to 35 ℃, and the mixture is stirred for 2 hours;
s13: diluting with water, adding H2O2Adding HCl to the solution to centrifugally wash the solution until the solution is yellow, and dialyzing the solution until the pH value is 6.0;
s14: centrifuging (3500rpm) for 10min, taking supernatant, and freeze-drying for 72h to obtain graphene oxide powder.
S2: and (5) placing the mixed solution obtained in the step (S1) in a micro injection pump connected with a high-voltage generator for electrostatic spinning to obtain the biological membrane.
Wherein the electrospinning parameters can be: the total concentration is 8-10%, the voltage is 10-15kV, the distance from a spray head to a receiver is 10-20cm, and the electrospinning flow rate is 1-3 mL/h. Preferably, these parameters are: the total concentration is 9 percent, the voltage is 12.5kV, the distance from a spray head to a receiver is 15cm, and the electrospinning flow rate is 2 mL/h.
And observing the appearance of the electrospun fiber by adopting a scanning electron microscope, and counting the average diameter and the diameter distribution of the fiber by using image J software. As shown in fig. 1 and fig. 2, the polycaprolactone/Gelatin/graphene oxide nanofiber composite membrane (PCL/Gelatin/GO membrane, i.e., the prepared biological membrane) prepared in this embodiment is in a porous fibrous shape, and the diameters of the nanofibers are different, mainly focusing on 200-400 nm.
Example two
This example examines the function and characteristics of the biofilm prepared in the first example.
(ii) induced differentiation of ESC-RPE cells
Continuously culturing human embryonic stem cell H1 (from the institute of biochemistry and cell biology of Shanghai Life sciences institute of Chinese academy of sciences, Cat. SCSP-301) for 35-45 days without passage, forming pigment focus with diameter of about 1mm, picking out pigment focus, inoculating into culture plate, collecting cells grown from pigment focus, namely RPE cells, when growing and fusing, digesting with 0.25% (mass percentage concentration) trypsin at 37 deg.C for 10min, counting, and counting with 1 × 105/cm2(ii) RPE cells were uniformly seeded onto cell culture plates coated with matrigel (BD Co., USA) and carefully transferred to a cell culture incubator at 37 ℃ with 5% CO2Culturing, and changing the liquid for half a day for 2-3 days.
The culture medium required for ESC-RPE cell culture was: high-sugar DMEM basal medium, 20% (mass percent concentration) KSR (KnockOut)TMSerum Replacement), 1% (mass percent concentration) of non-essential amino acids, 1mM L-glutamine, all available from Invitrogen, USA.
(II) construction of tissue engineered RPE graft
The biological membrane prepared in the first embodiment is cut into a circular sheet with the diameter of 10mm, the circular sheet is soaked in 75% alcohol for 30min, the circular sheet is rinsed for 2 times by PBS and then placed into a 48-hole plate, and each hole is inoculated with 1 × 105ESC-RPE cells were carefully transferred to a cell culture incubator at 37 ℃ with 5% CO2Culturing, and changing the liquid for half a day for 2-3 days.
The culture medium required for ESC-RPE cell culture was: high-sugar DMEM basal medium, 20% (mass percent concentration) KSR (KnockOut)TMSerum Replacement), 1% (mass percent concentration) of non-essential amino acids, 1mM L-glutamine, all available from Invitrogen, USA.
(III) detection of cell biological Properties of tissue engineered RPE
After ESC-RPE cells grow on a biological membrane for 14 days, the expression condition of RPE65 and ZO-1 is detected by an immunofluorescence method,
the method comprises the following specific steps: after 3 times of PBS washing, after 15 minutes of room temperature fixation with 4% paraformaldehyde, washing again with PBS for 3 times, adding 0.2% Trition X-100, after 30 minutes of room temperature treatment, washing with PBS for 3 times, sealing 5% goat serum for 1 hour at room temperature, diluting rabbit-derived ZO-1 antibody (Cat 13663S, Cell Signaling Technology, USA, dilution ratio 1:200) and mouse-derived RPE65 antibody (Cat 138ab 26, Abcam, UK, dilution ratio 1:200) with sealing solution, incubating overnight at 4 ℃, after 3 times of next day PBS washing, adding secondary antibody, incubating at 37 ℃ for 60 minutes, washing with PBS for 3 times, staining Cell nucleus for 10 minutes, washing cells with PBS for 3 times, adding fluorescence sealing solution, and observing under a fluorescence or confocal microscope.
The results are shown in FIG. 3, where the tissue engineered RPE is in a paving-stone-like morphology, expressing RPE65 and ZO-1.
(IV) detecting the ultrastructure of the tissue engineered RPE
ESC-RPE cells grow on a biological membrane for 30 days, and then the transmission electron microscopy is adopted to detect the ultrastructure of the RPE, and the method comprises the following specific steps: 2.5% glutaraldehyde overnight, after rinsing with PBS, after further fixation with 1% osmic acid for 2 hours, after rinsing with PBS, acetone was dehydrated in a gradient, embedded with epoxy resin 618, ultrathin sections were positioned, after uranium and lead staining, and observed by FEI company TECAI 10 transmission electron microscope.
As shown in FIG. 4, the tissue engineered RPE graft is a monolayer sheet, which forms a typical RPE morphology, the cell tip is rich in microvilli, pigment particles are concentrated above the cytoplasm, the nucleus is below the cytoplasm, the cells are in a monolayer, and tight junctions are formed between the connected cells.
(V) detecting the gene expression profile of the tissue engineered RPE
(1) Growing ESC-RPE cells on a biomembrane for 30 days, taking ESC-RPE cells cultured on a cell culture plate as a control, removing supernatant, washing with PBS for 3 times, adding 0.5mL of pancreatin, digesting at 37 ℃ for 5min, adding 1mL of culture medium for neutralization digestion, and centrifuging at 1500rpm at room temperature for 5 min;
(2) removing supernatant, adding 1mL Trizol (from Sigma), mixing cells, and standing at room temperature for 5 min;
(3) adding 0.2mL of chloroform, reversing for 15s, and standing at room temperature for 2-3 min;
(4)12000g at 4 ℃ centrifugal 15 min;
(5) sucking the upper aqueous phase and placing the upper aqueous phase in another EP tube;
(6) adding 0.5mL of isopropanol, reversing, and standing at room temperature for 10 min;
(7) centrifuging at 12000g for 10min at 4 deg.C;
(8) discarding the supernatant, and depositing RNA at the bottom of the tube;
(9) adding 1mL of 75% ethanol (suspension precipitation), and oscillating the centrifugal tube for a short time or slowly blowing and beating the precipitation by using a gun head;
(10) centrifuging at 7500g for 5min at 4 deg.C;
(11) discarding the upper 75% ethanol, and depositing RNA at the bottom of the tube;
(12) airing for 5-10 min at room temperature;
(13) with RNase-free ddH2O dissolved sample
(14) Preparing 1% agarose electrophoresis gel, adding 5 μ l total RNA sample into each sample loading hole, keeping constant voltage at 80V for 20min, taking out the gel, and analyzing bands by using a gel imaging system to detect RNA integrity;
(15) with RNase-free ddH2Using an ultraviolet spectrophotometer to measure the purity of the total RNA sample by taking O as a control, and ensuring that the ratio of OD260 to OD280 is 1.8-2.0;
(16) detecting the concentration of the total RNA sample by using a fluorescence quantitative instrument;
(17) the integrity of the total RNA sample is checked with a Bioanalyzer (e.g., Bioanalyzer 2100);
(18) by using
UltraTMRNA Library Prep Kit for
(NEB, USA) kit, cDNA library was constructed.
(19) Taking 3 mu g of total RNA of each sample, and purifying mRNA from the total RNA by adopting poly-T oligo-attached magnetic beads;
(20) preparing mRNA fragments with divalent cations in NEBNext First Strand Synthesis Reaction Buffer (5X);
(21) synthesizing a first single-stranded cDNA by using a random hexamer primer and M-MuLV reverse transcriptase (RNase H);
(22) synthesizing a second cDNA with DNA polymerase I and RNase H;
(23) converting the remaining extension fragments into blunt ends by exonuclease and polymerase;
(24) after adenylation of the 3' end of the DNA fragment, nucleic acid hybridization is performed with a NEBNext linker with a hairpin loop structure;
(25) AMPure XP system (Beckman Coulter, Beverly, USA) is adopted to purify the library, so that the length of the cDNA fragment is between 150 and 200 bp;
(26) adding 3 μ l USER enzyme (NEB, USA), and incubating at 37 deg.C for 15 min;
(27) incubating at 95 deg.C for 5 min;
(28) performing PCR reaction by using Phusion high-fidelity DNA polymerase and a universal PCR primer;
(29) PCR products were purified using AMPure XP system (Beckman Coulter, Beverly, USA) and library quality was assessed using an Agilent Bioanalyzer 2100 system;
(30) generating DNA clusters on a cBot Cluster Generation System with TruSeq PE Cluster kit (v3-cBot-HS, Illumia);
(31) sequencing the library by adopting an Illumina Hiseq 2500 platform;
(32) HTSeq v0.6.1 was used to calculate the number of reads per gene, the number of reads per megabase length (RPKM) from a gene per million reads was calculated, and the value of RPKM represents the gene expression level.
(33) The differential genes between the two groups were analyzed using a DESeq R package (1.10.1) and the resulting P-values were used to control the false positive rate using the Benjamini and Hochberg method. DESeq analysis P values < 0.05 were considered differentially expressed genes.
The results are shown in fig. 5 and 6, where the tissue engineered RPE has similar gene expression levels to the control group and the pearson correlation is greater than 0.98.
(VI) transplanting the polycaprolactone/gelatin/graphene oxide nanofiber composite membrane to the lower retinal cavity of rabbit eyes
Transplanting the polycaprolactone/gelatin/graphene oxide nanofiber composite membrane into the lower cavity of a rabbit eye retina, wherein the specific method comprises the following steps: taking 3-month-old New Zealand white rabbits, injecting ketamine (50mg/kg) and xylazine (10mg/kg) through muscles, dropping 2 drops of compound tropicamide eye drops through an operation eye, performing anesthesia and mydriasis satisfaction, performing a conventional 23G vitreous cutting operation, injecting about 40 mu L of sterile physiological saline into a subretinal space under direct vision to cause local retinal detachment, placing a polycaprolactone/gelatin/graphene oxide nanofiber composite membrane in a bullet shape of 1.1x2 mm into the subretinal space, and adding antibiotics continuously for five days after the operation.
(seventhly) Optical Coherence Tomography (OCT) detection of retina structure of rabbit eye after transplantation
3 days, 1 week and 4 weeks after surgery, animals were anesthetized routinely, mydriasis, retinal structure was examined using Heidelberg SD OCT, and scanning was performed using a 30 ° lens at a scanning position through the midline of the transplanted biomimetic membrane to examine retinal structure.
As shown in FIG. 7, the PCL/Gelatin/GO nanofiber composite membrane was successfully transplanted into the subretinal space, and the retina was partially detached in the operation area 3 days after the operation, but after 1 week, the retina was flattened, and the retina structure was well maintained after the PCL/Gelatin/GO nanofiber composite membrane was transplanted through 4 weeks of the operation.
(VIII) electrophysiological detection of retinal function of transplanted Rabbit eyes
After 4 weeks of operation, after general anesthesia of the animals, mydriasis, animal dark adaptation for 20min, retinal function was examined using the full-field electroretinogram diagnostic system (Espion, Diagnosis LLC). The specific method comprises the following steps: the cornea is dripped with the enoet gel to place a corneal contact lens electrode (ERG-jet), the reference electrode is arranged at the 5mm position of the caudal side of the outer eyelid margin at both sides under the skin, and the ground electrode is arranged at the middle part of the spinal column of the back under the skin. The dark adapted response wave was recorded after 1 minute using the clinical standard detection procedure. Both eyes were recorded simultaneously, and light adaptation was performed for 10 minutes after dark adaptation was completed, and then light adaptation response waves were recorded.
As shown in fig. 8, the PCL/Gelatin/GO nanofiber composite membrane had no significant effect on retinal function after transplantation compared to pre-operative and normal rabbit eyes.
(nine) histological method for detecting biocompatibility of transplanted graft
After 4 weeks post-surgery, both eyes were sacrificed and the anterior segment of the eye was removed after fixation overnight with 2% glutaraldehyde + 1.5% paraformaldehyde. After finding the graft area, the tissue was dissected in the retinal to scleral direction, and the graft area was taken about 1.5x2.5mm in size for embedding and toluidine blue staining of retinal sections.
As shown in FIG. 9, the retinal structure in the operative area remained good, and no significant retinal degeneration, apoptosis, etc. were observed.
Finally, the principle and embodiments of the present invention are explained by using specific examples, and the above descriptions of the examples are only used to help understand the core idea of the present invention, and the present invention can be modified and modified without departing from the principle of the present invention, and the modified and modified examples also fall into the protection scope of the present invention.
Claims (4)
1. A preparation method for constructing a biological membrane of tissue engineered retinal pigment epithelium is characterized by comprising the following steps:
s1: uniformly dissolving polycaprolactone, gelatin and graphene oxide in a polar solvent to obtain a mixed solution, wherein the mass percent concentrations of the polycaprolactone, the gelatin and the graphene oxide in the mixed solution are 69.6%, 29.8% and 0.6% respectively;
s2: and (5) placing the mixed solution obtained in the step (S1) in a micro injection pump connected with a high-voltage generator for electrostatic spinning to obtain the biological membrane.
2. The method of claim 1, wherein in step S1, the polar solvent is hexafluoroisopropanol.
3. The method of claim 1, wherein in step S1, the graphene oxide is in powder form and is prepared by the following steps:
s11: taking 10g of 32-mesh high-purity graphite, and adding the graphite into 500mL of concentrated sulfuric acid at 0 ℃;
s12: then 60g KMnO was added4Heating to 35 deg.C, and stirring;
s13: diluting with water, adding H2O2Adding HCl to the solution to centrifugally wash the solution until the solution is yellow, and dialyzing the solution until the pH value is 6.0;
s14: centrifuging for 10min, taking supernatant, and freeze-drying to obtain graphene oxide powder.
4. The method of claim 1, wherein in step S2, the electrospinning parameters are as follows: the total concentration is 8-10%, the voltage is 10-15kV, the distance from a spray head to a receiver is 10-20cm, and the electrospinning flow rate is 1-3 mL/h.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103656742A (en) * | 2013-11-29 | 2014-03-26 | 温州医科大学 | Preparation method of functionalized retinal pigment epithelial cell graft |
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